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Architecture of Mixed Calcium Oxalate Dihydrate and Monohydrate Stones

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Architecture of Mixed Calcium Oxalate Dihydrate and Monohydrate Stones Scanning Microscopy Volume 6 Number 1 Article 18 1-16-1992 Architecture of Mixed Calcium Oxalate Dihydrate and Monohydrate Stones Hidenobu Iwata Ehime University Shouzo Iio Ehime University Shunji Nishio Ehime University Masafumi Takeuchi Ehime University Follow this and additional works at: https://digitalcommons.usu.edu/microscopy Part of the Biology Commons Recommended Citation Iwata, Hidenobu; Iio, Shouzo; Nishio, Shunji; and Takeuchi, Masafumi (1992) "Architecture of Mixed Calcium Oxalate Dihydrate and Monohydrate Stones," Scanning Microscopy: Vol. 6 : No. 1 , Article 18. Available at: https://digitalcommons.usu.edu/microscopy/vol6/iss1/18 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Scanning Microscopy, Vol. 6, No. 1, 1992 (Pages 231-238) 0891- 7035/92$5. 00 +. 00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA ARCHITECTURE OF MIXED CALCIUM OXALATE DIHYDRATE AND MONOHYDRATESTONES Hidenobu Iwata*, Shouzo Iio, Shunji Nishio and Masafumi Takeuchi Department of Urology, Ehime University School of Medicine, Shigenobu, Ehime, 791-02 Japan (Received for publication May 7, 1991, and in revised form January 16, 1992) Abstract Introduction Calcium oxalate dihydrate (COD) and mono­ Calcium oxalate dihydrate (COD) and mono­ hydrate (COM) are the most frequent constituents hydrate (COM) are the most frequent constituents of urinary stones, and there still exist some of urinary stones. Therefore the effort of stone questions about the interrelation between the two research is mainly focused on the pathogenesis of hydrates. Architecture of mixed COD and COM calcium oxalate stones. Nevertheless many stones was observed by electron microscopy to questions remain unsolved. From the morpho­ solve the questions. logical point of view, the following points can The fractured surface of a stone is composed be given. of the fractured face of the crystals. In this 1. The dihydrate crystals are more situation a morphological criterion of typical frequently observed in the urinary sediment, dipyramid shape is useless to identify COD. But while in the stone both hydrates are equally we could identify COD using the partial dissolu­ often found (5, 7, 20). tion method, which etched square pits on COD 2. The crystal shape of dihydrate (octa­ crystals. hedral dipyramid) is essentially the same in the COD and COM formed distinctly separate urinary sediment and in the stone, but that of layers. COD was always found in the stone monohydrate is quite different (15, 25). surface and COM in the center. The stone surface 3. When both hydrates exist in a stone, was covered by a thick layer of organic matrix, the dihydrate is always found in the stone and the intercrystalline space was filled with surface and the monohydrate in the deeper layers matrix. The crystals were grown thrusting the (4, 18, 20). matrix aside to minimize the space. Recently scanning electron microscopy (SEM) Although COD is more soluble than COM, the has been widely used in stone research. Compared urine contains specific substances that favor the to polarization microscopy, SEM has some formation of COD. Supposing the stone matrix disadvantages since it is difficult to observe excludes these substances selectively, the gel­ the interior of the stones. In 1985 we reported state matrix provides a preferable condition for the partial dissolution method which allows the COM formation. This hypothesis is suitable to observation of the crystal arrangements and explain the high incidence of COM stones. An crystal-matrix interrelations on the fractured abrupt change of the crystalline constituent can surface of the stones (10). We later found that be explained by COD crystal deposition on COM this method etches surface marks which are stones. Frequent COD crystalluria can explain why characteristic of each crystalline constituent COD is always found in the stone surface. Once (12). The following studies were performed to the stone surface is covered with COD crystals, solve the questions described above using the they continue to grow in the gel-state matrix or partial dissolution method. deposit further to form the bulk of the stone. Materials and Methods Calcium oxalate dihydrate (COD) crystals Key Words: Calcium oxalate stone, Calcium oxalate COD crystals were obtained from the urine of dihydrate, Calcium oxalate monohydrate, Crystal a hypercalciuric patient. The crystal suspension dissolution, Organic matrix. was filtered through 0.22 pm Millipore filter, which was settled on the suction funnel. The crystals remained on the filter paper were washed *Address for correspondence: with pure water. Then several drops of 5%(wt/v) Hidenobu Iwata, tetrasodium ethylenediaminetetraacetate (EDTA) Department of Urology, Ehime University School of solution, adjusted to pH 7.2 with HCl, were Medicine, Shigenobu, Ehime, 791-02 Japan poured on the filter paper to start the Phone Number: 0899-64-5111 dissolution of the crystals. The dissolution was 231 H. Iwata, s. Iio, S. Nishio, M. Takeuchi Figure 1. SEM of COD crystals from the urine of a hypercalciuric patient. (a) Octahedral dipyramid shape of undissolved crystals. (b) Five-minute dissolution with a EDTA solution etched square pits on the surface of the crystals. The side of the square is always parallel to the base of the crystal pyramid. (c) In the early phase of dissolution the bottom of the pits is made up of four triangular planes. Bar = 5 µm. carried out at room temperat,,re, ;about 20' C. The fixative solution was exchanged for a dissolution dissolution was stopped after several minutes by solution, which was made by adding 5%(wt/v) EDTA washing with pure water. The filter papers were to the fixative mixture without phosphate buffer dried in a vacuum, mounted on aluminum stubs, and was adjusted to pH 7.2 with HCl. It took coated with platinum and observed with a Hitachi about two weeks for total dissolution of the S-500A scanning electron microscope. crystals with a daily change of the dissolution Calcium oxalate stones solution. Calcium oxalate stones selected for this The decrystallized specimens were further study were surgically removed from eight patients fixed in the fixative mixture for one day. The and were stored in our laboratory without any specimens were post-fixed in 1% osmium tetraoxide treatment for many years in the room atmosphere. in O.lM phosphate buffer, pH 7.2, for 4 hours. Each of these patients had multiple renal stones The post-fixed specimens were dehydrated with less than l cm in diameter and either a spheroid graded ethanol solutions and embedded in Epon- or mulberry shape, which are typical of calcium 812. Thin sections were cut and were double oxalate monohydrate (COM) stones. Five patients stained with uranyl acetate and lead citrate. had spheroid type stones and another three had Micrographs were taken with a Hitachi H-800 mulberry type stones. transmission electron microscope. Toluidine blue Partial dissolution method: Each stone was stained sections were examined by light micro­ fractured through its center with a razor blade. scopy ( LM). One fragment of each stone was immersed in the EDTA solution and another fragment was left Results untreated. The temperature of the dissolution solution was maintained at 20'C and dissolution Calcium oxalate dihydrate (COD) crystals time ranged from 5 to 30 minutes. Then the The COD crystals obtained from the urine of fragments were rinsed with pure water and dried a hypercalciuric patient showed typical in a vacuum. Both fragments of a stone were octahedral dipyramid of about 10-20 µm in size mounted on aluminum stubs, and further treated (Fig. la). A five-minute treatment with the EDTA for SEM as described above. solution etched square pits on the surface of the In order to observe the crystal-matrix crystals. The side of the square was always interface, transmission electron microscopy (TEM) parallel to the base of the crystal pyramid (Fig. was used. Some of the stone fragments were fixed lb). In the early phase of dissolution, the in a fixative mixture for about one week at room bottom of the pits was made up of four triangular temperature. The fixative mixture contained planes (Fig. le). These findings indicate that 5%(v/v) formalin, 2%(v/v) glutaraldehyde and COD crystals have an octahedral or dodecahedral 1%(wt/v) cetylpyridinium chloride in O.lM substructure. Thus the square etch pits can be phosphate buffer, pH 7.2 (10, 11). Then the used as another criterion to identify COD. 232 Architecture of Mixed Calcium Oxalate Stones Figure 2. SEM of fractured (a) spheroid stone and (b) mulberry shaped stone composed of COD and COM. The surface of these stones is rough, because it is filled with dipyramid COD crystals. A central core of one of the nodules is projecting on the fractured surface (white arrow). It is composed of a crystal aggregate. Bar= 500 pm. Figure 3. SEM of fractured surface of a COD stone. (a) Typical dipyramid shape of COD is seldom observed on the fractured surface. (b) Five-minute dissolution etched square pits on the fractured face of COD crystals. Bar= 5 pm. Calcium oxalate stones face of the crystals. Consequently, the typical Under the naked eye some of the stones were dipyramid shape was seldom seen (Fig. 3a). In lustered and others were lusterless. Under the this situation the partial dissolution method was scanning electron microscope the surface of the very useful. The dissolution solution etched former stones was smooth, and no crystal figure square pits on the fractured face of the crys­ was found. These stones were found to be tals, and we could identify such crystals to be composed of COMonly. But the surface of the COD (Fig.
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