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Molecular modifiers reveal a mechanism of pathological crystal growth inhibition

Abstract

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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Figure 1: Effect of inhibitors on COM crystallization.
Figure 2: Time-resolved imaging of COM crystal dissolution.
Figure 3: Mechanism and in silico study of inhibitor action.
Figure 4: In vitro assay and urinary excretion of HCA.

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Acknowledgements

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

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

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

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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.

Additional information

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 figures and tables

Extended Data Figure 1 Examples of ISE measurements.

In situ ISE measurements of COM crystallization in the presence of CA (a) and HCA (b) at concentrations of 0 μg ml−1, 20 μg ml−1, 40 μg ml−1, 60 μg ml−1, 80 μg ml−1 and 100 μg ml−1. The y axis is the quantity of free calcium ions in the growth solution that are consumed during crystallization. Linear regression of each curve provides the rate of crystal growth. The percentage inhibition of COM crystallization is obtained by comparing the slopes of ISE curves in the presence of inhibitor (filled symbols) to the slope in the absence of inhibitor (open diamonds). ppm, parts per million.

Extended Data Figure 2 Inhibitor speciation and its effect on COM growth.

Here we compare calculations of inhibitor speciation with ISE measurements of COM percentage inhibition at pH 6.2 (solid bars) and pH 8.0 (patterned bars). a, Percentage of deprotonated CA and HCA species, calculated from equations (4)–(9). Fully dissociated species (charge −3) are represented by white bars and partially dissociated species (charge −2) are represented by grey bars. b, Results of ISE measurements in supersaturated calcium oxalate solution (S = 3.8) in the presence of CCA = 20 μg ml−1 (orange bars) and CHCA = 20 μg ml−1 (blue bars). The percentage inhibition of COM crystal growth slightly decreases at higher alkalinity. HCA is the more effective inhibitor irrespective of solution pH. Data are the average of more than 10 measurements (error bars are 1 s.d.; P < 0.05 comparing HCA to CA at both levels of pH).

Extended Data Figure 3 Optical micrographs of COM crystals.

Optical micrographs of COM crystals after heating a growth solution for 3 days at 60 °C. Here we compare the control sample in the absence of growth inhibitor (a) to solutions prepared with CCA = 20 μg ml−1 (b) and CHCA = 20 μg ml−1 (c). Scale bars, 100 μm.

Extended Data Figure 4 HCA-induced etch pits on the COM (100) surface.

ac, Time-resolved images during in situ AFM measurements of a COM (100) crystal surface in the presence of HCA. The surface is first imaged in the absence of inhibitor (a) and then at CHCA = 0.25 μg ml−1 (b and c). The elapsed time between each deflection mode image is approximately 4 min. df, Height (or depth) profiles corresponding to the dashed lines in ac, respectively. As shown in a and d, the COM (100) surface before the addition of HCA is comprised of single steps with height approximately 0.4 nm (see inset in d), which approximately corresponds to the unit cell dimension in the [100] direction (a = 0.6 nm)48. Depth profiles in e and f show the temporal evolution of a single etch pit. Quantitative analysis of etch pit dimensions with respect to depth d (g) and width w (h) reveal monotonic changes during 10 min of continuous AFM imaging. Schematics of an etch pit (left inset in g) with highlighted depth (right inset in g) and height (inset in h) are shown to aid visualization.

Extended Data Figure 5 Construction of Bliznakov plots.

a, Theoretical Bliznakov plot for crystal inhibitors that follow a step-pinning mode of action as a function of increasing calcium oxalate relative supersaturation σ (derived from ref. 65). b, Plots generated from in situ AFM data on COM (010) surfaces compare changes in step velocity in the [] direction (purple squares) and [021] direction (blue diamonds) as a function of increasing CHCA. The deviation of experimental data from theoretical trends suggests that step pinning is not the dominant mechanism by which HCA inhibits COM surface growth.

Extended Data Figure 6 Binding energy of inhibitors on the COM (100) surface.

The results of DFT calculations showing the adsorption configuration and binding energy of CA3− (a), CA2− (b), HCA3− (c) and Ox2− (d) on the COM (100) surface and of HCA3− (e) and CA3− (f) binding to a (001) step on the COM (100) surface. For these calculations, the surfaces are kept frozen (that is, unrelaxed). Atoms are coloured as follows: hydrogen (white), carbon (grey), oxygen (red) and calcium (green).

Extended Data Figure 7 CA and HCA interactions with relaxed COM surfaces.

Superimposed structures of CA and HCA interacting with unrelaxed (coloured balls and sticks) and partially relaxed (yellow sticks) surfaces of COM crystals. Side-view snapshots depict CA interaction with (100) (a) and (021) (b) surfaces and HCA interaction with (100) (c) and (021) (d) surfaces. Atoms are coloured as follows: hydrogen (white), carbon (grey), oxygen (red) and calcium (green). The (100) surface is practically unaffected by the presence of HCA, whereas the (021) surface shows dislocations due to strain induced by the high binding affinity of the inhibitor (see also Extended Data Table 1). The total energy of the (100) face changes by +18.8 kcal mol−1 owing to HCA adsorption compared to the +28.1 kcal mol−1 energy change of the (021) face (positive signs are endothermic and energy values correspond to the difference between single point energy calculations of the COM surface with inhibitors removed). The corresponding values for the total energy change of the (100) and (021) faces from the presence of the CA are +14.2 kcal mol−1 and +25.7 kcal mol−1, respectively. The partial relaxation of COM surfaces compared to unrelaxed surface calculations (Extended Data Fig. 6) does not alter the overall trend in inhibitor–crystal binding affinity.

Extended Data Figure 8 Complexation of organic acids with calcium.

DFT-calculated binding energy (scaled per number of molecules, N) for the complexation of organic anions HCA3−, CA2− and Ox2− with calcium ions. Note that the data for HCA3− and Ox2− are identical to Fig. 3g, and are merely placed here for direct comparison with CA2−. Dashed lines connecting symbols are added to guide the eye.

Extended Data Table 1 Quantification of lattice strain in the presence of HCA and CA

Supplementary information

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. (MOV 792 kb)

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. (MOV 1030 kb)

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. (MOV 1032 kb)

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. (MOV 464 kb)

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. (MOV 6709 kb)

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Chung, J., Granja, I., Taylor, M. et al. Molecular modifiers reveal a mechanism of pathological crystal growth inhibition. Nature 536, 446–450 (2016). https://doi.org/10.1038/nature19062

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