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Natural Abundance 43Ca NMR as a Tool for Exploring Calcium Biomineralization: Renal Stone Formation and Growth Geoffrey M. Bowers*,† and R. James Kirkpatrick‡ † ‡
Division of Chemistry, Alfred University, 1 Saxon Drive, Alfred, New York 14802, United States College of Natural Science, Michigan State University, East Lansing, Michigan 48824, United States
bS Supporting Information ABSTRACT: Renal stone diseases are a global health issue with little effective therapeutic recourse aside from surgery and shockwave lithotripsy, primarily because the fundamental chemical mechanisms behind calcium biomineralization are poorly understood. In this work, we show that natural abundance 43Ca NMR at 21.1 T is an effective means to probe the molecular-level Ca2+ structure in oxalate-based kidney stones. We find that the 43Ca NMR resonance of an authentic oxalate-based kidney stone cannot be explained by a single pure phase of any common Ca2+-bearing stone mineral. Combined with XRD results, our findings suggest an altered calcium oxalate monohydrate-like Ca2+ coordination environment for some fraction of Ca2+ in our sample. The evidence is consistent with existing literature hypothesizing that nonoxalate organic material interacts directly with Ca2+ at stone surfaces and is the primary driver of renal stone aggregation and growth. Our findings show that 43Ca NMR spectroscopy may provide unique and crucial insight into the fundamental chemistry of kidney stone formation, growth, and the role organic molecules play in these processes.
R
enal stones are aggregates of microcrystalline biomineralized material that form in the kidneys and renal tubules of affected patients, causing severe pain and occasional renal failure.1 Despite the global occurrence of stone diseases,2,3 there are few effective therapeutic protocols to treat and prevent their formation. This is in large part due to a lack of knowledge regarding the fundamental chemical mechanism(s) underlying nucleation, aggregation, and growth of calcium biomineralizations in general, which include renal stones, arterial plaques,4,5 and biogenic amorphous calcium carbonates.6,7 The general chemical principles involved in calcium biomineralization and their relative importance in several systems are essential inputs for interdisciplinary teams seeking to understand crystallization on a fundamental level and those working to develop new biomaterials, synthetic methods, or therapies for biomineralization-related diseases. In turn, achieving a deeper understanding of the underlying chemistry and growth mechanisms requires the application of new tools to study biomineralization phenomena. In this work, we show that natural abundance 43Ca NMR holds great promise for advancing our molecular-scale understanding of Ca-biomineralization by testing the hypotheses that high-field (21.1 T) double frequency sweep (DFS)8,9 magic angle spinning (MAS) 43Ca NMR can distinguish between calcium oxalate-based renal stones and the common Ca2+-bearing minerals in these stones and that 43Ca NMR can provide direct evidence of molecular-scale Ca2+ organic interactions at stone surfaces. It is known that many renal stones consist primarily of calcium oxalate monohydrate (CaC2O4 3 H2O, whewellite) with lesser r 2011 American Chemical Society
amounts of calcium oxalate trihydrate (CaC2O4 3 3H2O). The other calcium oxalate hydrate commonly precipitated in the urinary tract, calcium oxalate dihydrate (CaC2O4 3 2H2O, weddellite), is also important, because most individuals who do not form stones pass calcium oxalate as very small dihydrate crystals without consequence. Of these phases, only calcium oxalate trihydrate has been studied previously using 43Ca NMR.10 It is also possible to find inclusions of calcium phosphate minerals in renal stones, with the most likely candidates being brushite (Ca(HPO4) 3 2H2O) or hydroxyapatite. Research has shown that Ca2+ interaction with organics, biomolecules, and tissue at oxalate stone surfaces contributes to stone growth,1 but a complete molecular-scale understanding of these organo Ca2+ interactions and the chemical mechanism by which they influence stone formation and growth remains largely unknown. Solid-state nuclear magnetic resonance (NMR) spectroscopy is a promising tool for exploring the microscale and molecularscale structure and dynamics of the chemical species and minerals involved in renal stones because it is the only analytical method that simultaneously probes both molecular-scale chemical interactions and microsecond to second time scale dynamics with elemental and site specificity. 13C and 31P NMR have already been used successfully to characterize the complex chemistry of renal stones,11,12 but 43Ca methods have not been used in this Received: September 17, 2011 Revised: October 31, 2011 Published: November 02, 2011 5188
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regard due to the unattractive NMR properties of 43Ca (γ = 2.87 vs 42.7 MHz/T for 1H; natural abundance = 0.145%) that lead to low sensitivity and lengthy acquisition periods. However, modern NMR techniques have been used to successfully study a number of important Ca2+-bearing materials without isotopic enrichment in the past decade,10,13 15 including several of the potential Ca2+ minerals in renal stones. Thus, it is reasonable to hypothesize that 43Ca NMR will prove effective for the as yet unstudied calcium oxalate monohydrate, calcium oxalate dihydrate, and an authentic oxalate-based renal stone. To test our hypotheses, 43Ca NMR experiments were performed on these three samples at 21.1 T in the High Field Magnetic Resonance User Facility at the Pacific Northwest National Laboratory (PNNL) using a facility-built 5 mm MAS
Figure 1. Ca coordination environments of CaC2O4 3 H2O (a) and CaC2O4 3 2H2O (b). Blocks c and d display the 43Ca DFS-MAS NMR spectra of CaC2O4 3 H2O and CaC2O4 3 2H2O, respectively (top), the best-fit line shape (middle), and the residuals (bottom).
probe. The CaC2O4 3 H2O was obtained from Alpha Aesar and used as-received. The CaC2O4 3 2H2O was synthesized according to the procedure of Doherty et al.16 Powder XRD shows that these samples are predominantly single phase and the nominal hydration states are stable over the length of our study (Figure S1 of the Supporting Information). Oxalatebased kidney stone samples were obtained from the Clement J. Zablocki VA Medical Center in Wisconsin. It was necessary to mix two of these samples to fill the MAS rotor, and XRD shows the mixed stone sample to be an excellent match for calcium oxalate monohydrate (Figure S4). We note that the conditions for the DFS preparatory pulses in the NMR experiments were carefully selected to avoid distorting the central transition and provided a signal-to-noise enhancement of 1.5 2.5 in 43Ca enriched calcium acetate and calcium carbonate (Figures S2 and S3). Greater detail regarding the experimental methods can be found in the Supporting Information. For CaC2O4 3 H2O, we observe a single broad resonance with no clearly discernible features above the noise, consistent with the known structural disorder of this phase (Figure 1). CaC2O4 3 H2O contains three H2O sites that are partially occupied with different probabilities,17 leading to a distribution of eight-coordinate Ca2+ environments in this material. Such disorder generally leads to a loss of resolvable features for a quadrupolar nucleus such as 43Ca and a Gaussian line shape, consistent with the high-quality fit obtained using a single Gaussian peak (Figure 1 and Table 1). Fitting this resonance with a single 1/2-integer (n/2) spin quadrupolar MAS line shape produces an ambiguous fit, as highlighted by the large uncertainty in the quadrupolar asymmetry parameter for an optimized fit involving ∼100 Hz of line broadening on the simulated spectrum (line 2 of Table 1). The average Ca O bond lengths for the various Ca2+ sites in CaC2O4 3 H2O are identical (Table 1), meaning that the isotropic chemical shifts for each Ca2+ coordination environment in the sample should be similar given the established relationship between the average Ca O bond distance and the 43Ca isotropic chemical shift.14,15 The Ca2+ sites differ from one another primarily in the standard deviation of the Ca O bond distances, suggesting a different local symmetry and unique set of quadrupolar parameters (and thus a unique second order quadrupolar shift) for each site. Because of the lack of spectral features, it was not possible to characterize the NMR parameters unambiguously on a site-bysite basis, although multiple field measurements18 and firstprinciples calculations may enable such an analysis in the future. CaC2O4 3 2H2O yields a quadrupolar powder pattern with a large quadrupolar asymmetry parameter (Figure 1), similar in appearance to the 43Ca NMR spectrum of CaC2O4 3 3H2O reported previously at lower field.10 The crystal structure of CaC2O4 3 2H2O is ordered and contains a single eight-coordinate Ca2+ site with two nearest neighbor H2O molecules.19
Table 1. Structural and NMR Parameters for the Three Ca Oxalate Hydrates and the Kidney Stonea sample
avg Ca O dist (Å) Ca O st dev (Å) CN δ (ppm) @ field fwhh (Hz) @ field 17
17
17
CaC2O4 3 H2O
2.44
CaC2O4 3 2H2O
2.4519
0.0419
819
2.4526
0.0526
826
CaC2O4 3 3H2O kidney stone
0.02
8
10.4 @21.1 T
fit type Gaussian
5.7 ( 0.8
2.5 ( 0.2
0.41 ( 0.30
Quad MAS
3.6 ( 1.8
2.5 ( 0.3
1.0
Quad MAS
4.2 ( 1.010 1.55 ( 0.110 0.72 ( 0.0510
553 ( 40 @21.1 T
Lorentzian 8.5 ( 0.6
a
η
CQ (MHz)
414 ( 40 @21.1 T
8.8 @14.1 T10 240 ( 40 @14.1 T 12.7 @ 21.1 T
δiso (ppm)
2.5 ( 0.4
0.75 ( 0.03
Quad MAS
CN = coordination number, fwhh = full width at half height. 5189
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Figure 2. Kidney stone 43Ca NMR spectrum (green) versus calcium oxalate monohydrate (red), calcium oxalate dihydrate (blue), and calcium oxalate trihydrate (black). Solid lines represent the experimental 43Ca NMR spectra collected as part of this work. Dashed lines are SIMPSON25 simulations of the associated experimental 43Ca NMR spectra. Note that only a simulation is presented for calcium oxalate trihydrate, as the 43Ca NMR spectrum for this material was published and characterized previously at a different applied magnetic field (ref 10).
The average Ca O bond distance is identical to that of CaC2O4 3 H2O, but with a larger standard deviation that we would expect to cause a more significant quadrupolar interaction. Indeed, within the signal-to-noise ratio limitations, the NMR resonance is well-fit with a single 1/2-integer (n/2) spin quadrupolar MAS powder pattern with a sizable quadrupolar interaction and statistical overlap with the monohydrate chemical shift (Table 1). While our data supports our initial hypothesis regarding the resolving power of high-field 43Ca NMR, we are unable to accept or reject the second component of our hypothesis (that 43Ca NMR can provide direct evidence of molecular-scale Ca2+ organic interactions at stone surfaces) on the basis of our results, though our combined XRD and 43Ca NMR data on the kidney stone are consistent with complexation of predominantly surface Ca2+ by nonoxalate organic molecules. The kidney stone 43Ca resonance is broad and featureless, encompassing a frequency region that overlaps with the full resonances for calcium oxalate monohydrate, brushite, and calcium citrate tetrahydrate (Figure 2 and Supporting Information Figure S5). This resonance is fairly well fit either with a single pure Lorentzian line shape or with a single high asymmetry 1/2 integer quadrupolar MAS line shape that includes a small amount of exponential apodization to represent structural disorder (Table 1). However, because the XRD results exhibit negligible variation from the ideal calcium oxalate monohydrate reference pattern, it is likely that our bulk material is pure calcium oxalate monohydrate. Therefore, our resonance is likely two highly overlapped peaks: one larger peak associated with the bulk ideal calcium oxalate monohydrate domains and the second due to an additional, smaller population of 43Ca nuclei that experience a different second order quadrupolar interaction and/or isotropic chemical shift. One potential explanation consistent with the XRD and 43Ca NMR data is that this second Ca2+ population represents surface Ca2+ on individual crystallites that experience coordination sphere distortions arising from partial coordination by deprotonated hydroxyl groups on proteins or small organics found in the urinary tract, leading to slightly altered NMR parameters. Preferential coordination of calcium by carboxylic functional groups on organic molecules has already been reported in molecular modeling of other aqueous systems,20,21 and nonoxalate organic material, particularly
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citrate, has been reported to exist between aggregated crystals in stones.1 This study demonstrates that it is possible to observe 43Ca at natural abundance in oxalate-based renal stones and that highfield 43Ca NMR can resolve differences between Ca2+ in stones and the common calcium-bearing minerals found in renal stones based primarily on variations in the 43Ca quadrupolar parameters. Our results are consistent with the idea that renal stones are aggregates of individual crystallites that form due to complexation of surface Ca2+ on multiple crystallites by carboxyl-bearing organics in the renal system. However, more detailed study of these materials involving multiple field measurements, doubleresonance techniques such as those recently applied to model 43 Ca-enriched biominerals by Wong et al.,22 or methods that can resolve phases based on differences in T123 will likely be a better test of whether these organo-Ca2+ interactions occur only at crystallite surfaces or as inclusions within crystallites. Such studies should also provide unique insight into the specific molecularlevel details of Ca2+-organo interactions and the biomineralization process, particularly when paired with molecular modeling.24 Ideally, the results of these studies will lead to development of effective therapeutic agents for treating stone diseases and industrial treatments for preventing fowling and mechanical failure due to organomediated aggregation of minerals.
’ ASSOCIATED CONTENT
bS
Supporting Information. XRD patterns, MAS and DFSMAS 43Ca spectra of the enriched model compounds, simulations of model phases and the renal stone at 21.1 T, enlarged images of Ca2+ coordination environments, and experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Telephone: 607-871-2822. E-mail:
[email protected].
’ ACKNOWLEDGMENT Funding for this research was provided by the United States Department of Energy, Grants DE-FG01-05ER05010201 and DE-FG02-10ER16128. The research was performed using EMSL, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research located at PNNL. Thanks to Dr. Niel Mandel of the Zablocki VA Medical Center for the kidney stone samples and to Drs. Andrew S. Lipton, Sarah Burton, and Pannusamy Nachimuthu for assistance with NMR or XRD experiments at PNNL. Thanks also to Dr. Jean Cardinale for access to her biosafety cabinet and to Swavek Zdzieszynski for helping acquire and analyze the kidney stone XRD pattern at Alfred University. ’ REFERENCES (1) Wesson, J. A.; Ward, M. D. Elements 2007, 3, 415–421. (2) Robertson, W. G. Urol. Res. 1990, 18, S3–S8. (3) Soucie, J. M.; Thun, M. J.; Coates, R. J.; McClellan, W.; Austin, H. Kidney Int. 1994, 46, 893–899. (4) Doherty, T. M.; Asotra, K.; Fitzpatrick, L. A.; Qiao, J.-H.; Wilkin, D. J.; Detrano, R. C.; Dunstan, C. R.; Shah, P. K.; Rajavashisth, T. B. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 11201–11206. 5190
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