纳米草酸钙的稳定性及其生长动力学研究
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摘要
泌尿系结石是一种世界范围的常见病、多发病。但至今为止,对尿石的预防和预前诊断尚无十分理想的方法,尿石症形成过程中的许多化学及物理问题还没有弄清。尿液中的纳米晶体是怎样生长聚集并最终形成结石?如何提高尿液中纳米晶体数量和尺寸检测的准确度?尿液浓缩导致草酸钙过饱和度增加,尿液中纳米晶体发生如何的变化?这些问题均不清楚。本文采用扫描电子显微镜(SEM)、纳米粒度仪(Nano-ZS)、光学显微镜、X-射线衍射分析(XRD)和紫外-可见吸收光谱(UV-vis)等方法,研究了纳米级草酸钙晶种性质,添加剂对尿液中纳米微晶稳定的影响,体外模拟了尿液放置及不同草酸钙过饱和度对尿液和纯水体系中沉降和悬浮纳米微晶的影响。
1.采用络合沉淀法合成了纳米草酸钙晶体,初步探讨了纳米草酸钙的性质。讨论了晶体浓度、超声时间、分散介质、添加剂、pH值等对纳米COD晶体分散和稳定性的影响；此外,研究了抑制剂(柠檬酸钾)与纳米COM和纳米COD晶体的相互作用及其差异。获得分散纳米二水草酸钙的优化条件；5×10-3mol／L的K3cit体系中纳米COM和COD微晶粒径最小且Zeta电位最负,最利于纳米COM和COD晶体的分散。
2.检测尿液中的纳米微晶,必须先将尿液过滤,以去除尿液中的微米级杂质。但由于过滤并超声后的尿微晶粒径很小,其表面能增加,尿纳米微晶容易团聚,因而导致尿微晶检测结果重现性不理想。通过加入不同添加剂(六偏磷酸钠、聚乙二醇4000(PEG4000)、PEG6000、十二烷基苯磺酸钠和十二烷基三甲基溴化铵(CTAB)),使尿液中纳米晶体达到稳定状态。结果表明,CTAB使得Zeta电位严重偏离尿微晶真实值,过滤并超声的尿液中加入PEG4000的粒径重复性最好,Zeta电位相似,利于粒径大小和Zeta电位的准确测定。
3.在正常人过滤尿体系中,随着放置时间的延长,体系中悬浮的晶体生长和聚集,沉降的晶体越来越多；纯水及尿液体系中加入不同过饱和度(RS)的草酸钙后,RS越高晶体沉降和生长的情况越明显。加入不同过饱和度草酸钙后,正常人尿液体系形成的晶体明显比水体系中小；但是随着尿液放置时间的延长,即使是在正常人尿液中,体系中悬浮的晶体生长和聚集,沉降的晶体越来越多,成石风险明显增大。
由于尿液中存在大量草酸钙微晶,本研究有助于进一步研究尿晶体生长、阐明草酸钙结石的形成机理和K3cit对草酸钙结石形成的抑制作用。
Urolithiasis is one of the most common urologic diseases in the world-wide area. However, up to date, there is no ideal method about the prevention of urinary stones and advanced diagnosis. The mechanism for the formation of urinary stones is not yet clearly understood and a number of questions about the physical and chemical factors still remain unanswered. How do these urinary nanocrystallites grow, aggregate, and form into urinary stones ultimately? How to improve the accuracy of detection of nanocrystallites in urine? How the changes of urinary nanocrystallites when urinary concentration increased the calcium oxalate supersaturation? The questions were not yet clearly understood. In this paper, the factors that affect property of calcium oxalate nanocrystallites, the effect of additives on the stabilization of nanocrystallites in urine, the settlement and suspension of nanocrystallites in the storage urine and in retention urine with different supersaturation of calcium oxalate were investigated using Scanning electron microscopy (SEM), Zetasizer Nano-ZS, optical microscopy, X-ray diffraction (XRD) and Ultraviolet-visible absorption (UV-vis). The results can be summarized as follows:
Firstly, the calcium oxalate nanocrystallites were prepared by complex precipitation method. The property of calcium oxalate nanocrystallites was discussed. The effects of concentration of crystallites, ultrasonic time, dispersed medium, additives and pH on the properties of COD nanocrystallites were investigated. In addition, the effects of inhibitors (potassium citrate) on the COM and COD nanocrystallites and the differents effects with K3cit between the two kinds of nanocrystallites were discussed. The best dispersion of COD nanocrystallites condition was got; When c(K3cit) was 5×10-3 mol/L, the COM and COD nanocrystallites had smallest size, the most negative Zeta value and best dispersion.
Secondly, in order to measure the nanocrystallites in the urine, the urine must be filtered firstly to remove micron impurities. However, as nanocrystal size is small, its surface free energy is higger, resulting in poor reproducibility of detection results. The differents additives (sodium hexametaphosphate, polyethylene glycol 4000 (PEG4000) and PEG6000, sodium dodecyl benzene sulfonate, dodecyl trimethyl ammonium bromides(CTAB)) can make the crystallites stability. The results showed that the CTAB can make the Zeta value seriouly diviate from the true value; the PEG4000, which was added in the urine, can make the best detection results (the best repeatability and similar Zeta value) and make accurate measurement of the size and Zeta value.
Thirdly, in filtrated health person urine, the suspended nanocrystallites were grow and aggregated; the sedimentation nanocrystallites were increased with the increased storage time. When the urine and pure water systerm was added in different supersaturations of calcium oxalate respectively, the higher the supersaturation degree of calcium oxalate it was, the more significantly deposition and growth of crystallites had. The crystallites in health person urine was smaller than that in pure water systerm. Even in health person urine, the suspended nanocrystallites were grow and aggregated; the sedimentation nanocrystallites were increased with the increased storage time; so the risk of stone formation was increased obviously.
Because there is a lot of calcium oxalate crystallites in urine, this study will help to further investigate the growth of urinary crystallites, clarify the formation mechanism of calcium oxalate stones and the inhibition of K3cit effect on stone formation.
1.采用络合沉淀法合成了纳米草酸钙晶体,初步探讨了纳米草酸钙的性质。讨论了晶体浓度、超声时间、分散介质、添加剂、pH值等对纳米COD晶体分散和稳定性的影响；此外,研究了抑制剂(柠檬酸钾)与纳米COM和纳米COD晶体的相互作用及其差异。获得分散纳米二水草酸钙的优化条件；5×10-3mol／L的K3cit体系中纳米COM和COD微晶粒径最小且Zeta电位最负,最利于纳米COM和COD晶体的分散。
2.检测尿液中的纳米微晶,必须先将尿液过滤,以去除尿液中的微米级杂质。但由于过滤并超声后的尿微晶粒径很小,其表面能增加,尿纳米微晶容易团聚,因而导致尿微晶检测结果重现性不理想。通过加入不同添加剂(六偏磷酸钠、聚乙二醇4000(PEG4000)、PEG6000、十二烷基苯磺酸钠和十二烷基三甲基溴化铵(CTAB)),使尿液中纳米晶体达到稳定状态。结果表明,CTAB使得Zeta电位严重偏离尿微晶真实值,过滤并超声的尿液中加入PEG4000的粒径重复性最好,Zeta电位相似,利于粒径大小和Zeta电位的准确测定。
3.在正常人过滤尿体系中,随着放置时间的延长,体系中悬浮的晶体生长和聚集,沉降的晶体越来越多；纯水及尿液体系中加入不同过饱和度(RS)的草酸钙后,RS越高晶体沉降和生长的情况越明显。加入不同过饱和度草酸钙后,正常人尿液体系形成的晶体明显比水体系中小；但是随着尿液放置时间的延长,即使是在正常人尿液中,体系中悬浮的晶体生长和聚集,沉降的晶体越来越多,成石风险明显增大。
由于尿液中存在大量草酸钙微晶,本研究有助于进一步研究尿晶体生长、阐明草酸钙结石的形成机理和K3cit对草酸钙结石形成的抑制作用。
Urolithiasis is one of the most common urologic diseases in the world-wide area. However, up to date, there is no ideal method about the prevention of urinary stones and advanced diagnosis. The mechanism for the formation of urinary stones is not yet clearly understood and a number of questions about the physical and chemical factors still remain unanswered. How do these urinary nanocrystallites grow, aggregate, and form into urinary stones ultimately? How to improve the accuracy of detection of nanocrystallites in urine? How the changes of urinary nanocrystallites when urinary concentration increased the calcium oxalate supersaturation? The questions were not yet clearly understood. In this paper, the factors that affect property of calcium oxalate nanocrystallites, the effect of additives on the stabilization of nanocrystallites in urine, the settlement and suspension of nanocrystallites in the storage urine and in retention urine with different supersaturation of calcium oxalate were investigated using Scanning electron microscopy (SEM), Zetasizer Nano-ZS, optical microscopy, X-ray diffraction (XRD) and Ultraviolet-visible absorption (UV-vis). The results can be summarized as follows:
Firstly, the calcium oxalate nanocrystallites were prepared by complex precipitation method. The property of calcium oxalate nanocrystallites was discussed. The effects of concentration of crystallites, ultrasonic time, dispersed medium, additives and pH on the properties of COD nanocrystallites were investigated. In addition, the effects of inhibitors (potassium citrate) on the COM and COD nanocrystallites and the differents effects with K3cit between the two kinds of nanocrystallites were discussed. The best dispersion of COD nanocrystallites condition was got; When c(K3cit) was 5×10-3 mol/L, the COM and COD nanocrystallites had smallest size, the most negative Zeta value and best dispersion.
Secondly, in order to measure the nanocrystallites in the urine, the urine must be filtered firstly to remove micron impurities. However, as nanocrystal size is small, its surface free energy is higger, resulting in poor reproducibility of detection results. The differents additives (sodium hexametaphosphate, polyethylene glycol 4000 (PEG4000) and PEG6000, sodium dodecyl benzene sulfonate, dodecyl trimethyl ammonium bromides(CTAB)) can make the crystallites stability. The results showed that the CTAB can make the Zeta value seriouly diviate from the true value; the PEG4000, which was added in the urine, can make the best detection results (the best repeatability and similar Zeta value) and make accurate measurement of the size and Zeta value.
Thirdly, in filtrated health person urine, the suspended nanocrystallites were grow and aggregated; the sedimentation nanocrystallites were increased with the increased storage time. When the urine and pure water systerm was added in different supersaturations of calcium oxalate respectively, the higher the supersaturation degree of calcium oxalate it was, the more significantly deposition and growth of crystallites had. The crystallites in health person urine was smaller than that in pure water systerm. Even in health person urine, the suspended nanocrystallites were grow and aggregated; the sedimentation nanocrystallites were increased with the increased storage time; so the risk of stone formation was increased obviously.
Because there is a lot of calcium oxalate crystallites in urine, this study will help to further investigate the growth of urinary crystallites, clarify the formation mechanism of calcium oxalate stones and the inhibition of K3cit effect on stone formation.
引文
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[20]Walton R C T, Kavanagh J P, Heywood B R, Rao P N. The association of different urinary proteins with calcium oxalate hydromorphs. Evidence for non-specific interactions. Biochim. Biophys. Acta,2005, 1723:175-183.
[1]Fragoso A C, Valla J S, Steyaert H, Arnaud P, Esposito C, Jose E C. Minimal access surgery in the management of pediatric urolithiasis. J. Pediatr. Urol.,2009,5:42-46.
[2]Takahiro Y, Masanori I, Sadao S, Kenjiro K. Prevalence and epidemiological characteristics of urolithiasis in Japan:national trends between 1965 and 2005. Urology,2008,71(2):209-213.
[3]Deng F, Ouyang J-M. Comparative investigations of ultrafine crystals in urine of healthy human and lithogenic patients. Mater. Sci. Eng. C,2006,26(4):688-691.
[4]Gui B-S, Huang Z-J, Xu X-J, Li M-R, He J-Y, Ouyang J-M. Measurement of urine crystallites and its influencing factors by nanoparticle size analyzer. J. Nanosci. Nanotechnol.,2010,10:5232-5241.
[5]许小晶,欧阳健明.邝荔,赵美霞.透射电子显微镜和激光散射对尿液中纳米微晶的研究.中华检验
医学杂志,2009,32(3)：330-332.
[6]雷琳,吕娜娜,吴明华,戚栋明.水相介质中纳米Ti02粒子软团聚体的解团聚.化工学报,2009,60(12)：3159-3164.
[7]Ren J, Song S. Valdivieso A L, Shen J, Lu S. Dispersion of silica fines in water-ethanol suspensions. J. Colloid Interface Sci.,2001,238:279-284.
[8]Lewis, Jennifer A. Colloidal processing of ceramics. J. Am. Ceram. Soc.,2000,83(10):2341-2359.
[9]Okubo T, Kiriyama. Structural and dynamic properties in the complex fluids of colloidal crystals, liquids and gases. J. Mol. Liq.,1997,72(1/3):347-364.
[10]Ruhlmann C, Thieme M, Helmstedt M. Interaction between dextran and human low density lipoproteins (LDL) observed using laser light scattering. Chem. Phy. Lipids,2001,110(2):173-181.
[11]徐康.用动态激光散射法测定悬浮液中颗粒尺寸分布的一些经验.分析测试技术与仪器,2006,12(2)：121-124.
[12]朱玉萍,王西奎,国伟林,王金刚.纳米银颗粒的制备及其影响因素研究.中国粉体技术,2009,25(6)：12-16.
[13]Pavhna V, Petr M. The influence of Cl-, SO42- and PO43- ions on the ζ-potential and microfiltration of titanium dioxide dispersions. Sep. Purif. Technol.,2007,58:295-298.
[1]邓耀良,覃光熙.草酸钙结石患者尿液中柠檬酸／草酸比值.中华实验外科杂志,1994,11(4)：205-206.尿液分析
[2]陈军浩,顾光煜,王以立.高锰酸钾褪色法测定尿液草酸.临床检验杂志,1999,17(5)：266-268.
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[5]Wang L J, Qiu S R, Zachowicz W, Guan X Y, James J. Yoreo D. Modulation of Calcium Oxalate Crystallization by Linear Aspartic Acid-Rich Peptides. Langmuir,2006,22:7279-7285.
[6]张利民.纳米颗粒分散过程机理研究.安徽电子信息职业技术学院学报,2005,4(6)：93-95.
[7]Wang L J, Zhang W, Qiu S R, Zachowicz W J, Guan X Y, Tang R, Hoyer J R, De Yoreo J J,Nancollas G H. Inhibition of calcium oxalate monohydrate crystallization by the combination of citrate and osteopontin. J. Cryst. Growth,2006,291(1):160-165
[8]Guerra A, Meschi T, Allegri F, Prati B, Nouvenne A, Fiaccadori E, Borghi L. Concentrated urine and diluted urine:the effects of citrate and magnesium on the crystallization of calcium oxalate induced in vitro by an oxalate load. Urol. Res.,2006,34(6):359-364.
[9]Wesson J A, Johnson R J, Mazzali M, Beshensky A M, Stietz S, Giachelli C, Liaw L, Alpers C E, Couser W G, Kleinman J G, Hughes J. Osteopontin is a critical inhibitor of calcium oxalate crystal formation and retention in renal tubules. J. Am. Soc. Nephrol.,2002,14(1):139-147.
[10]Ryall R L, Grover P K, Thurgood L A, Chauvet M C, Fleming, D E, Bronswijk W. The importance of a clean face:the effect of different washing procedures on the association of Tamm-Horsfall glycoprotein and other urinary proteins with calcium oxalate crystals. Urol. Res.,2007,35(1):1-14.
[11]何家扬,周任远,王文章,张燕宾.防石多糖对受损膀胱粘膜的保护作用.临床泌尿外科杂志,2000,15(11)：511-513.
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[1]Fragoso A C, Valla J S, Steyaert H, Arnaud P, Esposito C, Jose E C. Minimal access surgery in the management of pediatric urolithiasis. J. Pediatr. Urol.,2009,5:42-46.
[2]Takahiro Y, Masanori I, Sadao S, Kenjiro K. Prevalence and epidemiological characteristics of urolithiasis in Japan:national trends between 1965 and 2005. Urology,2008,71(2):209-213.
[3]Deng F, Ouyang J-M. Comparative investigations of ultrafine crystals in urine of healthy human and lithogenic patients. Mater. Sci. Eng. C,2006,26(4):688-691.
[4]Gui B-S, Huang Z-J, Xu X-J, Li M-R, He J-Y, Ouyang J-M. Measurement of urine crystallites and its influencing factors by nanoparticle size analyzer. J. Nanosci. Nanotechnol.,2010,10:5232-5241.
[5]许小晶,欧阳健明.邝荔,赵美霞.透射电子显微镜和激光散射对尿液中纳米微晶的研究.中华检验
医学杂志,2009,32(3)：330-332.
[6]雷琳,吕娜娜,吴明华,戚栋明.水相介质中纳米Ti02粒子软团聚体的解团聚.化工学报,2009,60(12)：3159-3164.
[7]Ren J, Song S. Valdivieso A L, Shen J, Lu S. Dispersion of silica fines in water-ethanol suspensions. J. Colloid Interface Sci.,2001,238:279-284.
[8]Lewis, Jennifer A. Colloidal processing of ceramics. J. Am. Ceram. Soc.,2000,83(10):2341-2359.
[9]Okubo T, Kiriyama. Structural and dynamic properties in the complex fluids of colloidal crystals, liquids and gases. J. Mol. Liq.,1997,72(1/3):347-364.
[10]Ruhlmann C, Thieme M, Helmstedt M. Interaction between dextran and human low density lipoproteins (LDL) observed using laser light scattering. Chem. Phy. Lipids,2001,110(2):173-181.
[11]徐康.用动态激光散射法测定悬浮液中颗粒尺寸分布的一些经验.分析测试技术与仪器,2006,12(2)：121-124.
[12]朱玉萍,王西奎,国伟林,王金刚.纳米银颗粒的制备及其影响因素研究.中国粉体技术,2009,25(6)：12-16.
[13]Pavhna V, Petr M. The influence of Cl-, SO42- and PO43- ions on the ζ-potential and microfiltration of titanium dioxide dispersions. Sep. Purif. Technol.,2007,58:295-298.
[1]邓耀良,覃光熙.草酸钙结石患者尿液中柠檬酸／草酸比值.中华实验外科杂志,1994,11(4)：205-206.尿液分析
[2]陈军浩,顾光煜,王以立.高锰酸钾褪色法测定尿液草酸.临床检验杂志,1999,17(5)：266-268.
[3]徐定邦,沙曼云,李应浦.有机酸发酵生产技术.化学工业出版社.1991,P133-137.
[4]Kavanagh J P. Supersaturation and renal precipitation:the key to stone formation? Urol. Res.,2006,34: 81-85.
[5]Wang L J, Qiu S R, Zachowicz W, Guan X Y, James J. Yoreo D. Modulation of Calcium Oxalate Crystallization by Linear Aspartic Acid-Rich Peptides. Langmuir,2006,22:7279-7285.
[6]张利民.纳米颗粒分散过程机理研究.安徽电子信息职业技术学院学报,2005,4(6)：93-95.
[7]Wang L J, Zhang W, Qiu S R, Zachowicz W J, Guan X Y, Tang R, Hoyer J R, De Yoreo J J,Nancollas G H. Inhibition of calcium oxalate monohydrate crystallization by the combination of citrate and osteopontin. J. Cryst. Growth,2006,291(1):160-165
[8]Guerra A, Meschi T, Allegri F, Prati B, Nouvenne A, Fiaccadori E, Borghi L. Concentrated urine and diluted urine:the effects of citrate and magnesium on the crystallization of calcium oxalate induced in vitro by an oxalate load. Urol. Res.,2006,34(6):359-364.
[9]Wesson J A, Johnson R J, Mazzali M, Beshensky A M, Stietz S, Giachelli C, Liaw L, Alpers C E, Couser W G, Kleinman J G, Hughes J. Osteopontin is a critical inhibitor of calcium oxalate crystal formation and retention in renal tubules. J. Am. Soc. Nephrol.,2002,14(1):139-147.
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