Molecular Medicine

, Volume 8, Issue 9, pp 525–535 | Cite as

The Effect of Preincubation of Seed Crystals of Uric Acid and Monosodium Urate with Undiluted Human Urine to Induce Precipitation of Calcium Oxalate in Vitro: Implications for Urinary Stone Formation

  • Phulwinder K. Grover
  • Rosemary L. Ryall
Original Articles



Previous studies demonstrated that crystals of uric acid (UA) and sodium urate (NaU) can induce the precipitation of calcium oxalate (CaOx) from its inorganic metastable solutions, but similar effects have not been unequivocally shown to occur in urine. The aim of this investigation was to determine whether preincubation of these seeds with urine alter their ability to induce deposition of CaOx from solution and thus provide a possible explanation for discrepancy of results obtained from aqueous inorganic solutions and undiluted urine.

Materials and Methods

The effects of commercial seed crystals of UA, NaU and CaOx (6 mg/100 ml) on CaOx crystallization were compared in a solution with the same crystals that had been preincubated for 3 hours with healthy male urine. A Coulter Counter was used to follow the crystallization and decrease in soluble 14C-oxalate was measured to determine the deposition of CaOx. The precipitated particles were examined by scanning electron microscopy (SEM). The preincubated seeds were demineralized and proteins released were analyzed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE).


Analysis of 14C-oxalate data revealed that while treated UA seeds did not affect CaOx deposition, those of NaU and CaOx inhibited the process by 51.9 (p ≤ 0.05) and 8.5% (p ≤ 0.05) relative to their respective untreated counterparts. Particle size analysis showed that the average modal sizes of particles precipitated in the presence of treated seed crystals of UA, NaU, and CaOx were very similar to those deposited in the presence of their respective untreated controls. These findings were confirmed by SEM which also showed that seed crystals of UA and NaU, untreated and treated, were attached like barnacles upon the surfaces of CaOx crystals which themselves were bigger than those precipitated in the presence of CaOx seeds. SDS-PAGE analysis of the demineralized treated seeds showed that they all selectively adsorbed urinary proteins, and perhaps other urinary macromolecules and low molecular weight components, on their surface.


It was concluded that preincubation with urine, such as occurs in vivo, only slightly reduces the ability of seed crystals of CaOx, but not of UA, to cause deposition of CaOx. The most striking effect was on NaU seeds where the preincubation quite dramatically attenuated their promotory effect on the mineral deposition. This may explain the discrepancy between findings of studies carried out in inorganic solutions and undiluted human urine. This stresses the invalidity of directly extrapolating results obtained in inorganic solutions to likely effects in urine and more importantly, on stone formation.


  1. 1.
    Ryall RL, Grover PK, Marshall VR. (1991) Urate and calcium stones: picking up a drop of mercury with one’s fingers? Am. J. Kidney Dis. 17: 426–430.CrossRefPubMedGoogle Scholar
  2. 2.
    Grover PK, Ryall RL. (1994) Urate and calcium oxalate stones: from repute to rhetoric to reality. Miner. Electrolyte Metab. 20: 361–370.PubMedGoogle Scholar
  3. 3.
    Grover PK, Ryall RL. (1995) Urate and calcium oxalate stones: A new look at an old controversy. In: Khan SR, (ed.) Calcium Oxalate in Biological Systems. CRC Press, New York and London; 305–321.Google Scholar
  4. 4.
    Coe FL, Raisen L. (1973) Allopurinol treatment of uric-acid disorders in calcium-stone formers. Lancet 1: 129–131.CrossRefPubMedGoogle Scholar
  5. 5.
    Coe FL, Kavalach AG. (1974) Hypercalciuria and hyperuricosuriain patients with calcium nephrolithiasis. N. Engl. J. Med. 291: 1344–1350.CrossRefPubMedGoogle Scholar
  6. 6.
    Smith MJV, Boyce WH. (1969) Allopurinol and urolithiasis. J. Urol. 102: 750–753.CrossRefPubMedGoogle Scholar
  7. 7.
    Pak CYC, Barilla DE, Holt K, et al. (1978) Effect of oral purine load and allopurinol on the crystallization of calcium salts in urine of patients with hyperuricosuric calcium urolithiasis. Am. J. Med. 65: 593–599.CrossRefPubMedGoogle Scholar
  8. 8.
    Miano L, Petta S, Gallucci M. (1979) Allopurinol in the prevention of calcium oxalate renal stones. Eur. Urol. 5: 229–232.CrossRefPubMedGoogle Scholar
  9. 9.
    Maschio G, Tessitore N, D’Angelo A, et al. (1981) Prevention of calcium nephrolithiasis with low dose thiazide, amiloride, and allopurinol. Am. J. Med. 71: 623–626.CrossRefPubMedGoogle Scholar
  10. 10.
    Smith MJV. (1983) Placebo versus allopurinol for recurrent urinary calculi. Proc. EDTA 20: 422–426.Google Scholar
  11. 11.
    Ettinger B, Tang A, Citron JT, et al. (1986) Randomized trial of allopurinol in the prevention of calcium oxalate calculi. N. Engl. J. Med. 315: 1386–1389.CrossRefPubMedGoogle Scholar
  12. 12.
    Royer L. (1928) Experimental research on parallel growth on mutual orientation of crystals of different species. Bull. Soc. Fr. Mineral. 51: 7–159.Google Scholar
  13. 13.
    Modlin M. (1967) The aetiology of renal stones: A new concept arising from studies in a stone-free population. Ann. R. Coll. Surg. Engl. (London) 40: 155–178.Google Scholar
  14. 14.
    Lonsdale K. (1968) Epitaxy as a growth factor in urinary calculi and gallstones. Nature 217: 56–58.CrossRefPubMedGoogle Scholar
  15. 15.
    Coe FL, Lawton RN, Goldstein RB, Tembe V. (1975) Sodium urate accelerates precipitation of calcium oxalate in vitro. Proc. Soc. Exp. Biol. Med. 149: 926–929.CrossRefPubMedGoogle Scholar
  16. 16.
    Pak CYC, Arnold LH. (1975) Heterogeneous nucleation of calcium oxalate by seeds of monosodium urate. Proc. Soc. Exp. Biol. Med. 149: 930–932.CrossRefPubMedGoogle Scholar
  17. 17.
    Meyer JL, Bergert JH, Smith LH. (1976) The epitaxially induced crystal growth of calcium oxalate by crystalline uric acid. Invest. Urol. 14: 115–119.PubMedGoogle Scholar
  18. 18.
    Koutsoukos PG, Lam-Erwin CY, Nancollas GH. (1980) Epitaxial considerations in urinary stone formation. I. The urateoxalate-phosphate system. Invest. Urol. 18: 178–184.PubMedGoogle Scholar
  19. 19.
    Mandel NS, Mandel GS. (1981) Epitaxis between stone-forming crystals at atomic level. In: Smith LH, Robertson WG and Finlayson B (eds.) Urolithiasis: Clinical and Basic Research. Plenum Press, New York and London, 469–480.CrossRefGoogle Scholar
  20. 20.
    Hartung R, Leskovar P, Kratzer M. (1981) Mechanisms of the influence of uric acid on the precipitation of Ca-oxalate crystals out of metastable solution. Croat. Chim. Acta 53: 381–388.Google Scholar
  21. 21.
    Deganello S, Coe F. (1983) Epitaxy between uric acid and whewellite: experimental verification. N. Jb. Miner. Mh. 6: 270–276.Google Scholar
  22. 22.
    Deganello S, Chou C. (1984) The uric acid-whewellite association in human kidney stones. Scan. Electron Microsc. II: 927–933.Google Scholar
  23. 23.
    Sarig S, Hirsch D, Garti N, Goldwasser B. (1984) An extension of the concept of epitaxial growth. J. Crystal Growth 69: 91–95.CrossRefGoogle Scholar
  24. 24.
    Tiselius HG. (1984) Effects of sodium urate and uric acid crystals on the crystallization of calcium oxalate. Urol. Res. 12: 11–15.CrossRefPubMedGoogle Scholar
  25. 25.
    Grases F, Conte A, Gil JJ. (1988) Simple method for the study of heterogeneous nucleation in calcium oxalate urolithiasis. Br. J. Urol. 61: 468–473.CrossRefPubMedGoogle Scholar
  26. 26.
    Sikiric M, Babic-Ivancic V, Tonkovic M. (1997) Precipitation of calcium oxalate and calcium phosphate in the presence of uric acid. Colloids Surfaces A: Physicochem. Eng. Aspects 121: 145–150.CrossRefGoogle Scholar
  27. 27.
    Grover PK, Ryall RL. (1997) Effect of seed crystals of uric acid and monosodium urate on the crystallization of calcium oxalate in undiluted human urinein vitro. Clin. Sci. 92: 205–213.CrossRefPubMedGoogle Scholar
  28. 28.
    Ryall RL. (1996) Glycosaminoglycans, proteins, and stone formation: adult themes and child’s play. Pediatr. Nephrol. 10: 656–666.CrossRefPubMedGoogle Scholar
  29. 29.
    Doyle IR, Ryall RL, Marshall VR. (1991) Inclusion of proteins into calcium oxalate crystals precipitated from human urine: a highly selective phenomenon. Clin. Chem. 37: 1589–1594.PubMedGoogle Scholar
  30. 30.
    Heukeshoven J, Dernick R. (1985) Simplified method for silver-staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6: 103–112.CrossRefGoogle Scholar
  31. 31.
    Stapleton AMF, Simpson RJ, Ryall RL. (1993) Crystal matrix protein is related to human prothrombin. Biochem. Biophys. Res. Commun. 195: 1199–1203.CrossRefPubMedGoogle Scholar
  32. 32.
    Stapleton AMF, Ryall RL. (1995) Blood coagulation proteins and urolithiasis are linked: crystal matrix protein is the F1 activation peptide of human prothrombin. Br. J. Urol. 75: 712–719.CrossRefPubMedGoogle Scholar
  33. 33.
    Dent CE, Sutor DJ. (1971) Presence or absence of an inhibitor of calcium oxalate crystal growth in urine of normals and stone-formers. Lancet ii: 775–778.CrossRefGoogle Scholar
  34. 34.
    Pak CYC, Hayashi Y, Arnold LH. (1976) Heterogeneous nucleation with urate, calcium phosphate and calcium oxalate. Proc. Soc. Exp. Biol. Med. 153: 83–87.CrossRefPubMedGoogle Scholar
  35. 35.
    Pak CYC, Holt K, Britton F, et al. (1980) Assessment of pathogenetic role of uric acid, monopotassium urate, monoammonium urate and monosodium urate in hyperuricosuric calcium oxalate nephrolithiasis. Miner. Electrolyte Metab. 4: 130–136.Google Scholar
  36. 36.
    Ryall RL. (1997) Urinary inhibitors of calcium oxalate crystallization and their potential role in stone formation. World J. Urol. 15: 155–164.CrossRefPubMedGoogle Scholar
  37. 37.
    Doyle IR, Ryall RL, Marshall VR. (1989) The effect of low speed centrifugation and millipore filtration on the urinary protein content. In: Walker VR, Sutton RAL, Cameron ECB, Pak CYC, Robertson WG (eds.) Urolithiasis. New York: Plenum Press; 593–594.CrossRefGoogle Scholar
  38. 38.
    Ryall RL, Harnett RM, Hibberd CM, et al. (1991) Effects of chondroitin sulphate, human serum albumin and Tamm-Horsfall mucoprotein on calcium oxalate crystallization in undiluted human urine. Urol. Res. 19: 181–188.CrossRefPubMedGoogle Scholar
  39. 39.
    Burns JR, Finlayson B. (1980) The effect of seed crystals on calcium oxalate nucleation. Invest. Urol. 18: 133–136.PubMedGoogle Scholar
  40. 40.
    Meyer JL. (1981) Nucleation kinetics in the calcium oxalate-sodium urate monohydrate system. Invest. Urol. 19: 197–201.PubMedGoogle Scholar
  41. 41.
    Iwata H, Kamei O, Abe Y, et al. (1988) The organic matrix of urinary uric acid crystals. J. Urol. 139: 607–610.CrossRefPubMedGoogle Scholar
  42. 42.
    Morse RM, Resnick MI. (1989) A study of the incorporation of urinary macromolecules onto crystals of different mineral compositions. J. Urol. 141: 641–644.CrossRefPubMedGoogle Scholar
  43. 43.
    Ryall RL, Grover PK, Stapleton AMF. (1995) The urinary F1 activation peptide of human prothrombin is a potent inhibitor of calcium oxalate crystallization in undiluted human urine in vitro. Clin. Sci. 89: 533–541.CrossRefPubMedGoogle Scholar
  44. 44.
    Grover PK, Ryall RL. (1999) Inhibition of calcium oxalate growth and aggregation by prothrombin and its fragments in vitro relationship between protein structure and inhibitory activity. Eur. J. Biochem. 263: 50–56.CrossRefPubMedGoogle Scholar
  45. 45.
    Burnier JP, Borowski M, Furie BC, Furie B. (1981) Gamma-carboxyglutamic acid. Mol. Cellular Biochem. 39: 191–199.CrossRefGoogle Scholar
  46. 46.
    Kalaiselvi P, Udayapriya KL, Selvam R. (1999) Uric acidbinding proteins in calcium oxalate stone formers and their effect on calcium oxalate crystallization. BJU Int. 83: 919–923.CrossRefPubMedGoogle Scholar
  47. 47.
    McCulloch RK, Bowyer RC, Brockis JW. (1981) Possible roles of urate in calcium urolithiasis. In: Brockis JG and Finlayson B (eds.) Urinary Calculus. PSG Publishing Company Inc., Littleton, Massachusetts; 347–352.Google Scholar
  48. 48.
    Grover PK, Ryall RL, Marshall VR. (1990) Effect of urate on calcium oxalate crystallization in human urine: evidence for a promotory role of hyperuricosuria in urolithiasis. Clin. Sci. 79: 9–15.CrossRefPubMedGoogle Scholar
  49. 49.
    Grover PK, Ryall RL, Marshall VR. (1992) Calcium oxalate crystallization in urine: role of urate and glycosaminoglycans. Kidney Int. 41: 149–154.CrossRefPubMedGoogle Scholar
  50. 50.
    Robertson WG, Knowles F, Peacock M. (1976) Urinary mucopolysaccharide inhibitors of calcium oxalate crystallization. In: Fleisch H, Robertson WG, Smith LH and Vahlensieck W (eds.) Urolithiasis Research. Plenum Press, New York and London; 331–334.CrossRefGoogle Scholar
  51. 51.
    Grover PK, Ryall RL, Marshall VR. (1993) Dissolved urate promotes calcium oxalate crystallization: epitaxy is not the cause. Clin. Sci. 85: 303–307.CrossRefPubMedGoogle Scholar
  52. 52.
    Kallistratos G, Timmermann A, Fenner O. (1970) Zum Einfluss des Aussalzeffektes auf die Bildung von calciumoxalat-Kristallen im menschlichen Harn. Naturwissenschaften 57: 198.CrossRefPubMedGoogle Scholar
  53. 53.
    Kallistratos G, Timmermann A. (1971) The “salting-out” effect as a possible causative factor for the formation of calcium oxalate crystals in human urine. Paper presented at the 66th annual meeting of the American Urological Association in Chicago, Illinois, USA, May 17, 1971, Urological Research Forum.Google Scholar

Copyright information

© NSLIJ Research Institute 2002

Authors and Affiliations

  1. 1.Division of Urology, Department of SurgeryFlinders University School of Medicine, Flinders Medical CentreBedford ParkAustralia

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