Letter | Published:

Molecular modifiers reveal a mechanism of pathological crystal growth inhibition

Nature volume 536, pages 446450 (25 August 2016) | Download Citation


Crystalline materials are crucial to the function of living organisms, in the shells of molluscs1,2,3, the matrix of bone4, the teeth of sea urchins5, and the exoskeletons of coccoliths6. However, pathological biomineralization can be an undesirable crystallization process associated with human diseases7,8,9. The crystal growth of biogenic, natural and synthetic materials may be regulated by the action of modifiers, most commonly inhibitors, which range from small ions and molecules10,11 to large macromolecules12. Inhibitors adsorb on crystal surfaces and impede the addition of solute, thereby reducing the rate of growth13,14. Complex inhibitor–crystal interactions in biomineralization are often not well elucidated15. Here we show that two molecular inhibitors of calcium oxalate monohydrate crystallization—citrate and hydroxycitrate—exhibit a mechanism that differs from classical theory in that inhibitor adsorption on crystal surfaces induces dissolution of the crystal under specific conditions rather than a reduced rate of crystal growth. This phenomenon occurs even in supersaturated solutions where inhibitor concentration is three orders of magnitude less than that of the solute. The results of bulk crystallization, in situ atomic force microscopy, and density functional theory studies are qualitatively consistent with a hypothesis that inhibitor–crystal interactions impart localized strain to the crystal lattice and that oxalate and calcium ions are released into solution to alleviate this strain. Calcium oxalate monohydrate is the principal component of human kidney stones16,17,18,19 and citrate is an often-used therapy20, but hydroxycitrate is not. For hydroxycitrate to function as a kidney stone treatment, it must be excreted in urine. We report that hydroxycitrate ingested by non-stone-forming humans at an often-recommended dose leads to substantial urinary excretion. In vitro assays using human urine reveal that the molecular modifier hydroxycitrate is as effective an inhibitor of nucleation of calcium oxalate monohydrate nucleation as is citrate. Our findings support exploration of the clinical potential of hydroxycitrate as an alternative treatment to citrate for kidney stones.

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J.D.R. acknowledges support from the National Science Foundation (grant 1207441) and the Welch Foundation (grant E-1794). G.M. acknowledges start-up funds from the University of Pittsburgh and computational support from the Center for Simulation and Modeling, and the Extreme Science and Engineering Discovery Environment, which is supported by the National Science Foundation (grant ACI-1053575).

Author information


  1. Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77204, USA

    • Jihae Chung
    •  & Jeffrey D. Rimer
  2. Litholink Corporation, Laboratory Corporation of America Holdings, Chicago, Illinois 60612, USA

    • Ignacio Granja
    •  & John R. Asplin
  3. Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA

    • Michael G. Taylor
    •  & Giannis Mpourmpakis


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J.C. performed data collection and analysis for bulk crystallization and in situ AFM studies, I.G. performed in vitro experiments in urine and analysed human trial samples, and M.G.T. performed DFT calculations. J.D.R. wrote the paper with help from G.M. and J.R.A., with all three authors contributing to the design and analysis of experiments. I.G. and M.G.T. contributed equally. All authors discussed the results and commented on the manuscript.

Competing interests

J.D.R. and J.R.A. have filed a provisional patent application on the use of organic acids as growth inhibitors of pathological calcification.

Corresponding authors

Correspondence to John R. Asplin or Jeffrey D. Rimer.

Reviewer Information Nature thanks J. Lieske, M. Sleutel and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information


  1. 1.

    COM (100) surface dissolution in the presence of CA

    Time-elapsed sequence of AFM deflection mode images depicting the growth of hillocks on a COM (100) surface in supersaturated CaOx solution (S = 4.1). Continuous imaging is initially performed in the absence of CA (time t = 0 to 3.8 minutes) followed by the addition of the same growth solution containing CCA = 0.10 μg/mL. The formation of etch pits occurs almost instantaneously upon introducing the inhibitor. The total imaging time for the in situ AFM video is 14.4 minutes.

  2. 2.

    COM (100) surface dissolution in the presence of HCA

    Time-elapsed sequence of AFM deflection mode images depicting the growth of hillocks on a COM (100) surface in supersaturated CaOx solution (S = 4.1). Continuous imaging is initially performed in the absence of HCA (time t = 0 to 6.7 minutes) followed by the addition of the same growth solution containing CHCA = 0.25 μg/mL. The formation of etch pits occurs almost instantaneously upon introducing the inhibitor. The total imaging time for the in situ AFM video is 32.7 minutes.

  3. 3.

    COM (010) surface dissolution in the presence of HCA

    Time-elapsed sequence of AFM deflection mode images depicting the growth of hillocks on a COM (010) surface in supersaturated CaOx solution (S = 4.1). Continuous imaging is initially performed in the absence of HCA (time t = 0 to 13.4 minutes) followed by the addition of the same growth solution containing CHCA = 0.10 μg/mL. The forward advancement of steps ceases upon introducing the inhibitor, and the steps recede (i.e., negative step velocity) toward the center of the screw dislocation with imaging time. Etch pit formation on terraces is observed during the course of surface dissolution at later times. The total imaging time for the in situ AFM video is 30.5 minutes.

  4. 4.

    COM (010) surface dissolution in undersaturated solution

    Time-elapsed sequence of AFM deflection mode images depicting the dissolution of hillocks on a COM (010) surface in undersaturated CaOx solution. Continuous imaging is initially performed in a supersaturated CaOx solution (S = 4.1) in the absence of inhibitor (not shown in the video). An undersaturated CaOx solution (S = 0.5) is introduced into the AFM liquid cell at time t = 0 minute. The forward advancement of steps ceases, and continuous imaging reveals that steps recede at a constant rate (i.e., negative step velocity) toward the center of the screw dislocation. Etch pit formation on terraces is also observed during the course of surface dissolution. The total imaging time for the in situ AFM video is 19.2 minutes.

  5. 5.

    Molecular conformations of inhibitor-calcium complexes

    Animation of geometry optimization during DFT calculations of two HCA molecules and two CA molecules in their fully deprotonated state (charge = –3) that form complexes with three Ca2+ cations. The total system is neutral in these calculations.

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