Indian Journal of Biochemistry & Biophysics Vol. 43, August 2006, pp. 226-232

Physical, X-ray diffraction and scanning electron microscopic studies of uroliths

Naveen Kumar*, Praveen Singh1+ and Satish Kumar2

Division of Surgery, 1Biophysics and Electron Microscopy Section, 2Central Instrumentation Facilities, National Biotechnology Center, Indian Veterinary Research Institute, Izatnagar (U.P.) 243 122, India

Received 2 January 2006; revised 15 May 2006

Identification of chemical constituents of calculus is important in the diagnosis and management of urolithiasis. The compositional variability of uroliths has different etiologies and requires various modes of treatment and prophylaxis. In the present study, we report the chemical compositional analyses of calculi recovered from buck and bullock by X-ray diffraction (XRD) and energy dispersive X-ray (EDX) techniques and ultra-structure examination by scanning electron microscopy (SEM). XRD and EDX investigations conclusively established the chemical compositions of urinary calculi under investigation. The calculus from buck (sample I) had calcium oxalate monohydrate, a dominant salt phase and magnesium compound in significant amount. The calculus from bullock (sample II) had magnesium ammonium phosphate phase, with significant amount of calcium in apatite form and K+ ions. SEM study at higher magnification (X1000) showed bipyramidal crystals in external zones of urolith (sample I). The struvite apatite calculus showed that basic unit of structure was lamination and the laminitis appeared to be made up of fine granules and high porosity. The bio-mineralization process of calculus formation was also studied, with a view to take preventive and therapeutic measures for amelioration of urinary stone diseases in animals and humans.

Keywords: Urinary calculi, Scanning electron microscopy, X-ray diffraction, Energy dispersive X-ray spectroscopy

Urinary stone disease affects large number of people and animals world-over1. Its prevalence has increased in the last two decades2. Urolithiasis can be caused by a range of medical and anatomical problems and often requires surgical intervention for management. Urinary calculi are the most common disease disorder, resulted from various factors such as metabolic abnormalities, nutritional factors, bacterial infection, and environmental conditions3-6. The most common forms of calcium crystals that precipitate in urine are calcium oxalate as whewellite (monohydrate) and weddllite (dihydrate) and calcium phosphate as hydroxyapatite (HAP), brushite and octacalcium phosphate. Most of human renal calculi

(approx 75%) are composed of calcium salts, primarily calcium oxalates and phosphates, and only about 10-12% are predominantly composed of uric acid7. Magnesium and calcium phosphates are reported to be the components of 25% urinary calculi8. Different constituents of urinary stones have different etiologies and require different mode of metaphylaxis and prophylaxis9. Studies on chemical composition and ultra-structure of various types of calculus can help understand the mechanisms of urolith nucleation, growth and aggregation and consequently help prevent the formation of stones or to dissolve stones, once formed in the urinary tract10,11.

X-ray diffraction (XRD) method has proved to be unambiguous, fast, sensitive and most reliable tool for structural analysis and segregation of phases of human/animal urinary calculi12,13. It is preferred over chemical methods for its complete identification of various crystallites present in calculus10,13. Energy dispersive X-ray (EDX) spectroscopy is employed to know the mineral elements in calculus. The scanning electron microscopic (SEM) studies help in understanding the morphology and ultra-structure of the calculi10,12,14. Application of these techniques in studying animals’ urinary calculi, particularly in

—————— *Author for correspondence Email: naveen@ivri.up.nic.in Phone: 0581-2302870 (O), 0581-2315128 (R) Fax: 91-581-2303284 +Present address: Ultra-Biology Lab, Department of Electronics Graduate School of Information Science and Electrical Engineering Kyushu University, Fukuoka, 812-8581, Japan Abbreviations: XRD, X-ray diffraction; EDX, energy dispersive X-ray; SEM, scanning electron microscopy; FTIR, fourier-transform infrared spectroscopy; HAP, hydroxyapatite; COM, calcium oxalate monohydrate.

KUMAR et al: ANALYSIS OF UROLITHS

227

ruminants may provide insight into understanding the physico-chemical process governing nucleation, growth and aggregation mechanisms. In the literature, only few reports5,10,11 are available on this aspect of research.

In the present paper, attempts have been made to analyze composition, structure and inner encrustation of urinary calculi obtained from the urethra of ruminants (a buck and bullock) using physical examination, X-ray diffraction, energy dispersive X-ray (EDX) and electron microscopy (EM) tools, with a view to unravel the mechanisms involved in the formation of urinary calculi.

Materials and Methods

The calculi recovered from two different clinical cases were subjected for detailed examination. Case 1 was a 2-year-old castrated male goat having complete urinary obstruction. Ultrasonographic examination of abdomen revealed full intact urinary bladder, whereas catheterization showed more than 20 golden-coloured small calculi lodged through out the urethra (Fig. 1, Sample I). Case 2 was a nine-year-old bullock having complete urinary obstruction since last 2 days. Rectal examination revealed tense, full intact urinary bladder. Prescrotal urethrotomy was performed and a single calculus was recovered at sigmoid flexure of penis (Fig. 1, Sample II).

Physical examination

The calculi recovered from the animals were washed with normal saline solution and later with distilled water. Visual observations were made of the external appearance, size, hardness and colour and after recording their physical characteristics, the calculi were split into 2-3 pieces with a hammer. They were subjected to X-ray diffraction (XRD) and energy dispersive X-ray (EDX) analysis and to scanning electron microscopic (SEM) studies.

X-ray diffraction

The powdered sample obtained by crushing the calculi in an agate mortor and pestle was used for XRD examination (Philips diffractometer model PW 1730/10) using Cu-Kα (λ = 1.54056 Å). Ni filter in wide range of Bragg’s angles 20° < 2θ < 70° at room temperature was used for carrying out XRD investigations. The operational parameters of X-ray spectrometer used in the study are given in Table 1.

Energy dispersive X-ray (EDX) analysis

Energy dispersive X-ray spectroscopy was employed on broken solid sample to obtain information on the mineral elements of calculi. Scanning electron microscope (LEO 440 fitted with EDX 7060 Oxford Instruments) was used for this study. DET area, 10 mm2; window, ATW 2; resolution at 5.9 KeV, 133 eV; and bias, -500 V were used for measurements. The instrument was calibrated against Co standard and operated at 20 kV.

Scanning electron microscopy (SEM) study

The calculi were washed with acetone, air-dried and mounted on aluminum stubs using carbon tape and sputtered with gold. The uroliths were observed under scanning electron microscope (JEOL-JSM-840, Japan). The texture of calculi were observed using back scattered electron (BSE) imaging15, first under low magnifications between 20 to 70X and subsequently under magnifications up to 4000X. Scanning electron microphotographs were taken to document the textures observed.

Results

Physical characteristics

The calculi recovered from case 1 were more than 20 in numbers having golden-beaded structure with

Fig. 1— Gross photograph of calculi of samples I and II, recovered from buck and bullock, respectively

Table 1—X-ray diffraction spectrometer’s operational parameters Parameters Value in units Radiation type, source X-rays Cu, 40 kV, 30 mA, Nickel

filtered

Wavelength used 1.540598 Å Kα1, monochromatic

λ Discriminator Diffracted beam, graphite-mono

λ Detector Scintillation

Temperature 22° ± 1

Range (2θ) 10-70°

Specimen motion None

Step size 0.02° 2θ

Scan rate 2°/min

INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, AUGUST 2006

228

smooth glistening surface. The size varied from 0.5 to 3 mm and appearance under light microscope was smooth. The urolith of case 2 had pyramidal structure, with dark gray rough dull surface having the size of 8.5 mm and showed cracked mass under light microscope.

XRD analysis

Analysis of urinary calculi by chemical methods is highly unsatisfactory. XRD technique identifies the calculus constituents by their unique diffraction patterns, which allow definite identification of unknown crystalline substances16. XRD analysis used to study the mineral components of calculi showed major peaks in the diffraction pattern, corresponding to the whewellite (Fig. 2a) and struvite (Fig. 2b).

Sample I calculus consisted of predominantly whewellite (CaC2O4.H2O) form of calcium oxalate monohydrate (COM). The reflection peaks, observed at around 16°, 30°, 48-50°, confirmed the presence of whewellite. The stone was dense and hard, spheroid in shape and showed pale-brown lamellae of whewellite. The high width of peaks in XRD pattern revealed nano-crystals of 100-125 Å embedded in organic matrix of calculi. Intensity of peaks was relatively less, as compared to struvite crystals, indicating lower crystallinity of oxalate calculus. Sample II calculus consisted mainly of struvite crystal as major mineral and HAP crystal as minor constituent. The reflection peaks observed around 15°, 45°, 46° and 47° confirmed the presence of struvite. Another phase of HAP in small quantity was also identified. The sharp, well-resolved peaks of calculi confirmed the bigger size crystals of phosphate mineral of approx 200-225 Å. The crystallinity in this calculus was higher as compared to oxalate crystals.

Crystal size was calculated from Scherrer’s equation17. The crystal size corresponding to each diffraction maxima could be determined from the measurement of the half width of diffraction peak. The data of crystal size corresponding to different peaks are shown in Table 2. The crystal size in struvite calculus was larger, as compared to oxalate calculus. The largest size obtained was of 220 Å, corresponding to HAP peak of mixed-type of calculus. Thus, bigger crystals were formed in rough-looking phosphate stones, whereas glistening, dense, solid appearance of oxalate calculi was due to the presence of smaller nano-size crystals and higher concentration of amorphous organic component in composite matrix. Table 3 shows XRD data of both the samples.

Fig. 2—X-ray diffraction patterns of (a) sample I (whewellite); (b) sample II (struvite) and (c) COM crystals with JCPDS International Centre data23

Table 2—XRD peaks and crystal size calculated from Scherrer’s equation

Types of stones

B (Half width in radians)

2θ (degree)

θ (degree)

Crystal size

(d) in Å Whewellite1

Whewellite2

Struvite1

Struvite2

Struvite3

HAP1

0.014

0.012

0.0078

0.0078

0.0087

0.0070

30.0

48.6

45.2

46.6

47.3

52.0

15.0

24.3

22.6

23.3

23.65

26.0

102.5

126.8

192.6

193.6

174.0

220.6

KUMAR et al: ANALYSIS OF UROLITHS

229

Energy dispersive X-ray (EDX) analysis

EDX data of both samples are given in Table 4. Element percent was the weight percent of the element after correction for inter-element effects.

Element % = Apparent conc./Intensity correction … (1)

Atomic percent was calculated from the measured weight percent values. For each element, it was weight percent/atomic weight; the sum of atomic weights for all elements in the sample was then normalized to 100%. Phosphorous element was present in insignificant amount in sample I. The

crystal phase was predominantly oxalate phase of calcium. Mg2+ ions were also present, but not in oxalate form. EDX data of sample II showed that the calculus was a predominantly magnesium phosphate stone, along with calcium apatite crystals, as observed in XRD pattern also. EDX analyses of samples I and II vindicated the results obtained from XRD analyses. SEM study

Both uroliths differed morphologically and constituently under SEM observation. Under relatively low magnification (X20), urolith of case 1 showed smooth-beaded appearance (Fig. 3a) and on

Table 3—XRD peaks and crystal size of urinary calculi (case 1 and 2)

Sample I (case 1)

2θ (degree)

θ (degree)

D (Å)

Rel. Int. (I/Io × 100)*

30.0 48.6 49.0

15.0 24.3 24.5

2.9761 1.8718 1.8574

100 98 85

Sample II (case 2)

15.0 19.2 45.3 46.4 47.2

7.5 9.6

22.65 23.2 23.6

5.9013 4.6188 2.0067 1.9553 1.9240

77 64

100 86 66

*Relative X-ray intensity on percent ratio, I0 is intensity of maximal peak

Table 4—EDX analysis of sample I (case 1) and II (case 2)

Case 1

Element Spectrum type

Element %

Atomic %

No. of ions

C Mg P

Ca O

Total

K ED K ED K ED K ED

18.35 3.62 0.18

18.84 59.01

100.00

26.15 2.55 0.10 8.05

63.15 100.00

13.25 1.29 0.05 4.08

32.00 Cation Sum

18.67

Case 2 Mg P N Ca O

Total

K ED K ED K ED K ED

20.83 26.30 2.10 1.90

48.86 100.00

17.63 17.47 1.11 0.98

62.82 100.00

8.98 8.90 0.56 0.50 32

Cation sum 18.94

KED represents the K spectrum line energy in atom, high difference in energy value, so more reliable as compared to L line

Fig. 3—SEM photographs of uroliths of sample I (a) showing beaded appearance, X20; (b) on slightly higher magnification X60; (c) on higher magnification (X1000) showing amorphous deposition and pitted surface; (d) showing multi-layered deposition (X500); and (e) showing bipyramidal crystals on further magnification, X1000

INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, AUGUST 2006

230

slightly higher magnification (X60), not much difference was observed (Fig. 3b). However, on higher magnification (X1000), surface of urolith appeared roughened and amorphous deposition and pitted surface was seen (Fig. 3c). Broken urolith showed multi-layered depositions, which were more clearly visible at higher magnification (X500) (Fig. 3d). On further magnification (X1000), external zones of a urolith showed bipyramidal crystals (Fig. 3e).

The urolith of case 2 showed well-defined central nucleus (nidus) under SEM observation. On lower magnification (X25) (Fig. 4a), nidus appeared homogenous, surrounded by well-defined laminations of uneven thickness, which on cross-section had stacked parallel rows. Thickness of laminae increased from center towards periphery. On higher magnification (X500), laminations were more clearly visible (Fig. 4b) and there was difference in porosity of different layers. The centre and intermediate zone showed coarse granular morphology and high porosity. The porosity decreased as the development of fine, dense concentric laminations proceeded towards the urolith surface. The struvite apatite calculus showed that basic unit of structure was lamination and the laminitis appeared to be made up of fine granules and high porosity. At the exposed interfaces of laminations, the surface was either

smooth or had projections of various shapes and craters or pores of various sized (Fig. 4c). The irregular deposits of apatite in between the laminae were observed. The fine concentric laminations composed of struvite formed the framework, whereas deposition of apatite occurred as amorphous fine patches between struvite crystals or surrounded struvite crystals (Fig. 4d). Localized differences in the crystalline or amorphous contents could be seen, but the overall composition was also the same in all the layers.

Discussion The results of present study showed identification

of chemical constituents and ultra-structure of calculi. The X-ray diffractograms of calculi were indexed with X-ray powder diffraction pattern of standard whewellite and struvite and also confirmed with earlier studies18-21. The pattern exhibited diffraction lines superposed on a broad hump extending from 10° to 22°, indicating the amorphous nature of examined calculi sample. Other diffraction lines were produced due to crystalline phases. In sample I, crystalline phase identified was due to COM. The reflection peaks observed at around 16°, 30°, 48-50° confirmed the presence of COM (whewellite). In XRD patterns, main diffraction peaks were located at 2.97 Å and 1.87 Å, which correspond to (2 0 2) and (3 0 3)

Fig. 4 —SEM photographs of uroliths of sample II (a) showing well-defined nucleus (nidus), X25; (b) showing laminations on higher magnification, X500; (c) showing pitted roughened surface, X250; and (d) showing fine laminations and deposition of apatite, X500

KUMAR et al: ANALYSIS OF UROLITHS

231

crystal planes of COM, respectively. Other crystal planes such as (1 0 1), prominent otherwise, were not grown much, presumably due to the presence of carbonate organic matrix around the crystals. The whewellite stone is the most thermodynamically stable and prevalent form of stone. Calcium oxalate is found in monohydrate and dihydrate form in urinary stone and could be distinguished from XRD pattern and FTIR spectra of calculi14,18. The broad spectral lines in oxalate crystals were due to nano-sized crystalline particles.

XRD pattern of sample II displayed a large number of peaks, positions of which in general matched to those stoichiometric struvite MgNH4PO4.6H2O

22,23. Nevertheless, many peaks were shifted and there was redistribution of their intensities, as compared to the ASTM reference23. This may be caused by the presence of other isomorphic mixtures of cations in the mineral, resulting in distortion in crystal lattice.

EDX analyses of both the calculi proved to be very useful in identification of elements present and their ratio. EDX studies corroborated the finding of XRD, and revealed the presence of Mg2+ ions in sample I, presumably in carbonate form, which was not observed in XRD analyses. Similarly, sample II was found to be a mixed-type stone with magnesium ammonium phosphate (struvite) phase along with calcium apatite crystal.

SEM is a powerful tool for the diagnosis of surface morphology of various substances, including calculi. It is the most suitable technique for study of crystal growth, detection of organic matrix and minor components of calculi24. A calculus may be defined as poly-crystalline concretion, found in the urinary tract and containing primary organic or inorganic crystalloids and much smaller amount of organic matrix25. The pathogenesis of stone formation remains poorly understood, but there are three major theories concerning their development26: (i) precipitation-crystallization theory incriminates supersaturation of urine with crystalloids as the primary factor in precipitation and subsequent growth of calculi, (ii) matrix-nucleation theory implies that some organic matrix substance in urine are responsible for the initial development of stone, and (iii) inhibition absence theory suggests that the absence of some critical inhibitors of stone formation is the primary factor in stone formation.

Crystallization of solids from an aqueous solution can be classified into two processes, nucleation and

crystal growth. A critical nucleus is formed during nucleation, followed by crystal growth and these two processes may continue until equilibrium is attained. The ease of nucleation is increased with increasing supersaturation27. Presence of bacteria in urinary passages is a predisposing factor in the formation of calculi. The occurrence of struvite in animals as well as human urinary stones has been taken as indicative of infection by urea-splitting organisms.

Formation and growth of urinary calculi involves the precipitation and accretion of crystals from the urine. Calculi grow through accretion of spherule aggregates. When fewer sphericules are present, growth proceeds through precipitation of fine radiating crystals (cement) in the form of bands. More commonly, growth occurs through a combination of two processes28. The continuity of bands indicates contemporaneous formation and similarity of chemical composition. Presence of pores, open spaces and fissures in between major laminations shows the changes in urine composition and interruption of the growth of calculi29.

Precipitation and dissolution of calculi are spontaneous processes that can take place simultaneously within an individual calculus30. The crystals that precipitate reflect the chemistry and composition of urine, from which they are formed. Because the composition of urine changes over the time, so can the composition of crystals that precipitate. Pre-existing crystals can dissolve, when compositional differences develop between these earlier formed crystals and the medium (urine) that surrounds them. However, this can only take place, if there are passageways that allow urine to circulate through the calculus, so that urine can interact with the crystals. The primary pores are avenues for the flow of urine through the calculus. These pores are original features directly related to the accretion of sphericules and are a direct consequence of the geometry and packing of sphericules, band or cements28. Porosity development begins with these primary features, which are inherent to the growth or accretion of a calculus. Primary porosity allows access of urine throughout the calculus. This, in turn, can lead to development of secondary porosity30.

Urinary calculi in goat and cattle commonly consist of calcium, ammonium and magnesium carbonate and phosphate, but silicones and oxalate containing calculi have also been reported31. Although, their etiology is not fully understood, there appears to be a number of

INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, AUGUST 2006

232

factors involved in the formation of calculi10. It is thought that diet and alkaline urine may predispose to urinary calculus formation. The role of diet rich in the formation of calcium carbonate calculi has been reported in ruminants32,33. Feeding of high phosphate diet to ruminants could result in the formation of urinary calculi containing calcium31, whereas low phosphorous/normal calcium diet could produce calculi in weanling rats34. Ruminants feeding on pasture low in phosphorous have shown an increased rate of formation of calcium oxalate and calcium carbonate calculi35. This may be due to the formation of an organic matrix, rather than through the direct effect on mineral metabolism.

Struvite and HAP are less soluble in alkaline urine. It is thought that the increase in urinary pH and liberation of ammonia from urea by bacterial urease reduce the solubility of struvite and provide ammonium (NH4

+) ions for precipitation25. Struvite crystals are sensitive to the degree of supersaturation and variations in growth rates of struvite result in changes of crystal habit36. The factors promoting struvite precipitation are Mg2+ ion supersaturation, presence of NH4

+ and PO4-3 ions and high urinary pH

or alkaline environment37,38. It is also found that struvite can precipitate in acidic sterile human urine between pH 5.63 and 7.037. As the solubility of struvite is pH-dependent, supersaturation with respect to struvite depends on initial concentrations and initial pH of the solution39.

References 1 Grases F, Costa-Bauza A & Garcia-Ferragut L (1998) Adv

Colloid Interface Sci 74,169-179 2 Trinchieri A, Coppi F, Montanari E, Del Nero A, Zanetti G

& Pisani E (2000) Eur Urol 37, 23-32 3 Seftel A & Resnick M I (1990) Urol Clin North Am 17, 159-

169 4 Lifshitz D A, Shalhav A L, Lingeman J E & Evan A P

(1999) J Endourol 13, 669-678 5 Hesse A & Heimbach D (1999) World J Urol 17, 308-315 6 Trinchieri A, Castelnuovo C, Lizzano R & Zanetti G (2005)

Urol Res 33, 194-198 7 Coe F L, Parks J H & Asplin J R (1992) N Engl J Med 327,

1141 8 Mandel G, Mandel N (1996) Analysis of stones in Kidney

stones (Coe F L, Favus M J, Pak C Y C, Parks J H & Preminger G M, eds), pp 323, Lippincott-Raven, Philadelphia

9 Rodgers A L, Mezzabotta M, Mulder K J & Nassimbani L R (1981) J South Afr Vet Assoc 52, 139-142

10 Wang X, Huang K, Gao J, Shan X, Lin C & Zhang G (1997) Res Vet Sci 62, 275-280

11 Ulrich L K, Kathleen A B, Koehler L A & Swanson L (1996) Vet Clin North Am: Small Ani Pract 26, 393-400

12 Prien E (1963) J Urol 89, 917-926 13 Wang X, Canfield P L & Gallagher C H (1985) Res Vet Sci

39, 373-377 14 Escolar E & Ballanato J (2003) Vet Rec 152, 625-628 15 DeNee P B & Abraham J L (1974) in Principles and

Techniques of Scanning Electron Microscopy (Hayay M A, ed), pp. 144-180, Van Nostrand Reinhold Co., New York

16 Hukins D W L (1981) X-ray diffraction by disordered

and ordered systems, pp. 129-138, Pergamon Press, New York

17 Culity B D (1967) Elements of X-ray Diffraction, 2nd edn, pp. 99, Addison-Wesley, MA

18 Krausova D (1996) Bull Czech Slovak Crystallogr Assoc 3(2), 148-150

19 Sinha A, Ingle A, Munim K R, Vaidya S N, Sharma B P & Bhisey A N (2001) Bull Mater Sci 24, 653-657

20 Quyang J M, Chen D Z& Zhong J P, (2004) Mol Crys Liq

cryst 420, 91-96 21 Shall H E I, Jcon J H, Abdel-Aal E A, Khan S, Gower L &

Rabinovich Y (2004) Cryst Res Technol 39, 214-221 22 Gibson R I (1974) Am Mineral 59, 1177-1186 23 Powder Diffraction File (1973) Joint Committee on Powder

Diffraction Standards Easton, ASTM file No. 15-762 24 Kim K M (1982) Scan Electron Microsc 4, 1635-1660 25 Moreau P M (1990) Vet Rec 125, 415-425 26 Osborne C A, Olover J E & Polzin D E (1980) in Current

Veterinary Therapy (Kirk R W, ed), VII edn, pp 1128, W B Saunders & CO., Philadelphia

27 Berner R A (1981) in Kinetics of weathering and diagenesis (Lasaga A C & Kirkpatric R T, eds), Vol. 8, pp. 111-134, Minerological Society of America, Washington D C

28 Neumann R D, Ruby A L, Ling G V, Schiffman P & Johnson D L (1994) Am J Vet Res 55, 1357-1367

29 Neumann R D, Ruby A L, Ling G V, Schiffman P & Johnson D L (1996) Am J Vet Res 57, 12-24

30 Bathurst R G C (1975) Carbonate sediments and their

diagenesis, pp. 231-338, Elsevier Scientific Publishing Co., New York

31 Blood D C, Radostits O M, Henderson J A, Arundal J H & Gay C C (1983) in Text Book of Veterinary Medicine, pp. 360-366, Bailliere Tindall, London

32 Talapatra S K, Ray S C & Sen K C (1948) J Agric Sci 48, 169-173

33 Sutherland A K (1958) Aust Vet J 34, 44-46 34 Coburn S P & Packett Jr L V (1962) J Nutr 76, 385-392 35 McIntosh C H, Pulsford M F, Spender W G & Rosser H

(1974) Aust Vet J 50, 345-352 36 Abbona F & Boistelle R (1979) J Cryst Growth 46, 339-354 37 Boistelle R, Abbona F & Berland Y (1984) in International

Symposium on Urolithiasis and Related Clinical Research (Schwille P O, Smith L H, Robertson W G et al., eds), pp. 793-797, Plenum Press, New York

38 McLean R J C, Downey J & Clapham I (1990) Urol Res 18, 39-43

39 Abbona F, Lundager E, Madsen H E & Boistelle R (1982) J Cryst Growth 57, 6-14