Role of Lipids in Urinary Stones: Studies of Calcium Oxalate

Calcium oxalate is the principal mineral component of most urinary stones. Membrane constituents associate either actively or passively with calcific ...
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InVited Feature Article Role of Lipids in Urinary Stones: Studies of Calcium Oxalate Precipitation at Phospholipid Langmuir Monolayers Daniel R. Talham,*,† Renal Backov,† Isa O. Benitez,† Denise M. Sharbaugh,† Scott Whipps,† and Saeed R. Khan‡ Department of Chemistry, UniVersity of Florida, GainesVille, Florida 32611-7200, and Department of Pathology, UniVersity of Florida, GainesVille, Flordia 32610 ReceiVed September 13, 2005. In Final Form: NoVember 18, 2005 This article reviews the authors’ experiments on calcium oxalate growth at lipid monolayers. Calcium oxalate is the principal mineral component of most urinary stones. Membrane constituents associate either actively or passively with calcific minerals during stone formation, and it has been proposed that lipid assemblies play a significant role, possibly providing sites for the initial nucleation event. Langmuir monolayers allow systematic studies of the heterogeneous precipitation of calcium oxalate at lipid assemblies. The influences of the chemical identity of the lipid headgroup, the organization of the monolayer, and the presence of heterogeneities and phase boundaries within the monolayer have been explored.

Introduction Kidney stones afflict patients worldwide. In the United States, alone, kidney stones account for over one million hospital admissions per year, and 12% of men and 4.5% of women will develop a stone by the age of 70.1 Stones can be deposits of calcium phosphates, uric acid, struvite, or even calcium carbonate, but by far the most common are those that contain calcium oxalate as the principal inorganic component. The crystalline inorganic material is always mixed with an organic matrix that accounts for about 2-3% of the total mass and is comprised of proteins, carbohydrates, lipids, and other cellular components (Figure 1). Oxalate and phosphate are concentrated in the kidney resulting in supersaturation with respect to their calcium salts, but the solution is metastable, meaning that heterogeneous nucleation is the predominant precipitation process. Calculations show that urinary concentrations and the rate of fluid flow in the kidney provide insufficient transit time for crystals to grow large enough to be occluded and retained.2 Indeed, crystals formed in the urinary tract of most humans are harmlessly excreted with the aid of protein and small molecule inhibitors. Specific urinary substances such as citrate, glycosaminoglycans, and the proteins osteopontin, bikunin, and CAI (crystal adhesion inhibitor) are thought to aid the process.3-5 However, in some cases, the crystals remain inside the kidneys and initiate the process of stone formation. Crystal attachment to the kidney’s tubular cell surface is therefore a critical step in pathological calcification, and it is thought that * To whom correspondence should be addressed. E-mail: talham@ chem.ufl.edu. † Department of Chemistry. ‡ Department of Pathology. (1) Soucie, J. M.; Coates, R. J.; McClellan, W.; Austin, H.; Thun, M. Am. J. Epidemiol. 1996, 143, 487. (2) Finlayson, B.; Reid, F. InVest. Urol. 1978, 15, 442. (3) Khan, S. R.; Kok, D. J. Front. Biosci. 2004, 9, 1450. (4) Ebisuno, S.; Nishihata, M.; Inagaki, T.; Umehara, M.; Kohjimoto, Y. J. Am. Soc. Nephrol. 1999, 10, S436. (5) Kumar, V.; Yu, S. H.; Farell, G.; Toback, F. G.; Lieske, J. C. Am. J. Physiol.-Renal 2004, 287, F373.

cell injury can provide sites for crystal nucleation, aggregation, and retention within the kidneys.6-15 In addition, cell death and degradation results in the production of membrane vesicles,6,8 which can further facilitate heterogeneous crystal nucleation and aggregation.16 Tissue culture studies have provided insights into renal responses to oxalate exposure. LLC-PK1 cells are commonly used to represent the proximal tubular cells and MDCK cells to represent epithelial cells of the more distal sections of the renal tubules. Renal epithelial cells in culture are injured by exposure to high levels of oxalate or calcium oxalate crystals,7-9,17 and epithelial injury has been shown to promote attachment of CaOx crystals.10,11 Functional cultures of MDCK cells are largely protected against calcium oxalate crystal adherence, but epithelial injury and the subsequent wound healing lead to increased crystal binding.10 Tissue injury can cause loss of cell polarity or membrane lipid asymmetry leading to changes in the composition and physical properties of the plasma membrane that alter crystalmembrane interactions.13 A variety of anionic cell surface (6) Hackett, R. L.; Shevock, P. N.; Khan, S. R. J. Urology. 1990, 144, 1535. (7) Hackett, R. L.; Shevock, P. N.; Khan, S. R. Urol. Res. 1994, 22, 197. (8) Khan, S. R.; Byer, K. J.; Thamilselvan, S.; Hackett, R. L.; McCormack, W. T.; Benson, N. A.; Vaughn, K. L.; Erdos, G. W. J. Am. Soc. Nephrol. 1999, 10, S457. (9) Miller, C.; Kennington, L.; Cooney, R.; Kohjimoto, Y.; Cao, L. C.; Honeyman, T.; Pullman, J.; Jonassen, J.; Scheid, C. Toxicol. Pharmacol. 2000, 162, 132. (10) Verkoelen, C. F.; van der Boom, B. G.; Houtsmuller, A. B.; Schroder, F. H.; Romijn, J. C. Am. J. Physiol. 1998, 274, F958. (11) Mandel, N. J. Am. Soc. Neph. 1994, 5, S 37 (12) Bigelow, M. W.; Wiessner, J. H.; Kleinman, J. G.; Mandel, N. S. J. Urol. 1996, 155, 1094. (13) Weissner, J. H.; Hasegawa, A. T.; Hung, L. Y.; Mandel, G. S.; Mandel, N. S. Kidney Int. 2001, 59, 637. (14) Lieske, J. C.; Leonard, R.; Swift, H.; Toback, F. G. Am. J. Physiol.-Renal 1996, 39, F192. (15) Lieske, J. C.; Swift, H.; Martin, T.; Patterson, B.; Toback, F. G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 6987. (16) Fasano, J. M.; Khan, S. R. Kidney Int. 2001, 59, 169. (17) Scheid, C.; Koul, H.; Hill, W. A.; LuberNarod, J.; Jonassen, J.; Honeyman, T.; Kennington, L.; Kohli, R.; Hodapp, J.; Ayvazian, P.; Menon, M. J. Urology. 1996, 155, 1112.

10.1021/la052503u CCC: $33.50 © 2006 American Chemical Society Published on Web 01/19/2006

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have all been shown to initiate calcium phosphate precipitation in vitro in metastable solutions.20,21 To better understand the role of lipids in urinary stone formation, we have undertaken studies of the heterogeneous precipitation of calcium oxalate at lipid assemblies. For this, we use Langmuir monolayers as models for lipid membranes or other cellular lipid structures. Monolayers of phospholipids are frequently used as models to investigate the physical and interfacial properties of biological membranes.22-24 A principal benefit is that the composition and physical state of the lipid interface can be controlled and analyzed. Studies of biomineral nucleation and growth at organized organic surfaces is also an established application of Langmuir monolayers.25-27 Heywood and Mann26,27 introduced studies on the growth of calcite and other particles at Langmuir monolayers. Landau et al.28,29 showed that oriented growth of glycine and NaCl takes place at the air/ water interface of Langmuir monolayers, including those formed from amino acids. These early studies showed that geometric, electrostatic, or stereochemical relationships between the crystal and the organic interface play important roles in polymorph or crystal face selectivity of the inorganic mineral. In this article, we review our own experiments of calcium oxalate growth at lipid monolayers. Studies have been performed to explore the influence of the chemical identity of the lipid headgroup, the organization of the monolayer, and the presence of heterogeneities and phase boundaries within the monolayer. Through this work, we have developed a better understanding of the mechanism of calcium oxalate precipitation at lipid interfaces. At the same time, even though we are drawn to investigate calcium oxalate because of its pathological impact, the system also provides insight into the more general question of the role of organic interfaces in purposeful biomineralization. Experimental Setup Figure 1. a. Calcium oxalate urinary stone, approximately 1.5 cm across. b. TEM image of a calcium oxalate crystal embedded in cellular debris after demineralization, fixation with malachite green aldehyde, and osmium tetroxide staining. The dumbbell-shaped object in the center is the calcium oxalate ghost. The darkly stained organic matrix associated with the crystal is clearly seen.

molecules, which mediate crystal attachment, can be exposed during cell proliferation or injury.13 It is clear that membrane constituents associate either actively or passively with calcific minerals during stone formation, and it has been proposed that lipid assemblies play a significant role, including providing sites for the initial nucleation event.12,18 Calcium oxalate crystal attachment is mediated by oxalate-induced exposure of phosphatidylserine (PS) on cell surfaces.11-13 Enrichment of cell membranes with PS resulted in increased attachment of CaOx crystals,11 whereas incubating cells with a PS-specific binding protein decreased crystal attachment.13 Furthermore, membrane vesicles, isolated from epithelial cells of rat kidney, catalyze calcium oxalate precipitation from metastable solutions.16 In a related study, organic matrix isolated from urinary stones also induced calcium oxalate precipitation.19 In fact, it was found that lipids from the stone matrix appear to be better nucleators than the complex matrix. Cell membrane lipids have also been implicated in nucleation of calcium phosphate crystals. Acidic phospholipids, lipid extracts from calcified crystals, membranes of matrix vesicles, and liposomes (18) Khan, S. R.; Atmani, F.; Glenton, P.; Hou, Z.-C.; Talham, D. R.; Khurshid, M. Calcif. Tissue Int. 1996, 59, 357. (19) Khan, S. R.; Shevock, P. N.; Hackett, R. L. J. Urol. 1988, 139, 418.

A scheme of the experimental setup appears in Figure 2. The state of the lipid monolayer is routinely monitored by correlating surface pressure measurements and Brewster angle microscopy. The composition of the aqueous subphase is controlled, and for crystal growth experiments, the Ca2+ and oxalate2- concentrations are metastable with respect to calcium oxalate, typically RS ) 5 or RS ) 10 (RS, relative supersaturation, is the ratio of the chemical activity of the solution to the equilibrium activity, including the influence of other ions). Calcium oxalate crystals growing at the surface are detected as very bright spots in Brewster angle microscopy,30,31 which is sensitive to only the interface proving that the crystals grow at the monolayer. This point is backed up by control experiments with and without the lipid monolayer.32,33 Crystal formation is (20) Anderson, H. C. Path. Ann. 1980, 15, 45. (21) Eanes, E. D.; Hailer, A. W. Calcif. Tissue Int. 1985, 37, 390. (22) Heckl, W. M.; Losche, M.; Scheer, H.; Mohwald, H. Biochim. Biophys. Acta 1985, 810, 73. (23) McConnell, H. M.; Watts, T. H.; Weiss, R. M.; Brian, A. A. Biochim. Biophys. Acta 1986, 864, 95. (24) Takao, Y.; Yamauchi, H.; Manosroi, J.; Abe, M. Langmuir 1995, 11, 912. (25) Mann, S.; Heywood, B. R.; Rajam, S.; Birchall, J. D. Nature 1988, 334, 692. (26) Heywood, B. R.; Mann, S. J. Am. Chem. Soc. 1992, 114, 4681. (27) Mann, S.; Archibald, D. D.; Didymus, J. M.; Douglas, T.; Heywood, B. R.; Meldrum, F. C.; Reeves, N. J. Science 1993, 261, 1286. (28) Landau, E. M.; Levanon, M.; Leiserowitz, L.; Lahav, M.; Sagiv, J. Nature 1985, 318, 353. (29) Landau, E. M.; Grayer Wolf, S.; Levanon, M.; Leiserowitz, L.; Lahav, M.; Sagiv, J. J. Am. Chem. Soc. 1989, 111, 1436. (30) Benitez, I. O.; Backov, R.; Khan, S. R.; Talham, D. R. Mater. Res. Soc. Symp. Proc. 2003, 774, 209. (31) Benitez, I. O.; Talham, D. R. Langmuir 2004, 20, 8287. (32) Whipps, S.; Khan, S. R.; O’Palko, F. J.; Backov, R.; Talham, D. R. J. Cryst. Growth 1998, 192, 243. (33) Letellier, S. R.; Lochhead, M. J.; Cambell, A. A.; Vogel, V. Biochim. Biophys. Acta-Gen. Subjects 1998, 1380, 31.

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Figure 2. Scheme of the experimental setup for studying calcium oxalate precipitation at Langmuir monolayers surrounded by figures of representative data. a. Crystals forming at the Langmuir monolayer are seen as very bright spots by Brewster angle microscopy (BAM). b. SEM image of crystals and monolayer removed from the Langmuir trough. c. The characteristic shape of a calcium oxalate crystal grown at a phospholipid monolayer is shown in a close-up SEM image. The six-sided (1h01) face is most commonly oriented parallel to the lipid interface. d. AFM image of a sphyngomyelin/dihydrocholesterol rich lipid raft and surrounding POPC transferred onto mica. e. Brewster angle microscope image of a DPPC Langmuir monolayer held in the LE/LC coexistence region. The lighter areas are the LC phase and the darker areas the LE phase. quantified by combining in situ BAM observations with ex situ electron microscopy and diffraction.

Heterogeneous Precipitation: Influence of the Lipid Interface A series of dipalmitoylphosphatidyl lipids with choline, glycerol, and serine headgroups (Chart 1) was initially used to demonstrate that precipitation of calcium oxalate at lipid interfaces can be effectively studied at Langmuir monolayers.32,34 At these lipid interfaces, calcium oxalate precipitates as the monohydrate (COM, also called whewellite),35 the form that predominates in urinary stones and in other model studies with lipid-containing organic matrix. The crystals also exhibit a high degree of selectivity in crystal habit and orientation, with the COM (1h01) face presented at the lipid interface (Figure 2c). For each of the monolayers studied, over 90% of the observed single crystals orient in this way. In urinary stones, COM forms as collections of tabular crystals. Flattened crystals with large (1h01) faces are often observed when surface modifiers are present,36 and this crystal face has previously been implicated in studies of COM precipitation and adhesion to membranes in lipid enriched cell culture studies.11,12 Recent AFM studies have shown that small molecule and biopolymer additives bind strongly to this face, altering crystal habit and rate of growth.37,38 Electrostatic arguments are used to account for the highly specific crystal orientation that is observed. The [1h01] direction consists of alternating layers of positive and negative net charge, so a polar face is generated, assuming the ionic arrangement of the bulk structure (Figure 3).35,36 The calcium rich layer has a slight positive charge, and two of the normally eight Ca2+ coordination sites in the bulk are empty on the crystal face. Therefore, calcium-binding anionic species greatly stabilize (34) Backov, R.; Khan, S. R.; Mingotaud, C.; Byer, K.; Lee, C. M.; Talham, D. R. J. Am. Soc. Nephrol. 1999, 10, S359. (35) Deganello, S. Acta Crystallogr. 1980, B37, 826. (36) Millan, A. Cryst. Growth Des. 2001, 1, 245.

this face. Each of the lipids we studied is known to bind Ca2+, implying a mechanism whereby Ca2+ is concentrated at the interface and the (1h01) face is stabilized by adsorption of the lipids. The structure of the surfactant headgroup also influences COM precipitation in support of this mechanism. Crystallization is enhanced at the anionic DPPG monolayer relative to either the zwitterionic DPPS or DPPC interfaces.32,34,39

Does the Interface “Template” the Crystal? It is generally accepted that organic media in biomineralization serve to concentrate and sequester ions as well as to direct the placement and structure of the inorganic salt.40 To direct structure, the regulatory interactions between the organic media and the inorganic solid likely include electrostatic effects coupled with favorable stereochemical or geometric relationship between the molecular arrangements of the surfaces forming the interface.40 At the time of our early experiments, it was attractive to think of the organic interface providing a template with epitaxial match for the inorganic surface, but whether templating truly takes place, and if so, the extent to which such a template is static or dynamic was in question. We designed a set of experiments41 to probe the effects of the packing density and packing geometry of the lipids within the monolayer and its compressibility on heterogeneous COM precipitation. Keeping with the glycerol headgroup, which was shown to effectively nucleate COM, we chose four amphiphiles with different lipophilic tails, (DPPG, DMPG, POPG, and DOPG) shown in Chart 1. Each behaves (37) Sheng, X. X.; Jung, T. S.; Wesson, J. A.; Ward, M. D. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 267. (38) Jung, T.; Sheng, X. X.; Choi, C. K.; Kim, W. S.; Wesson, J. A.; Ward, M. D. Langmuir 2004, 20, 8587. (39) Khan, S. R.; Glenton, P. A.; Backov, R.; Talham, D. R. Kidney Int. 2002, 62, 2062. (40) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: New York, 2001. (41) Backov, R.; Lee, C. M.; Khan, S. R.; Mingotaud, C.; Fanucci, G. E.; Talham, D. R. Langmuir 2000, 16, 6013.

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Chart 1

Figure 3. Calcium oxalate monohydrate crystal structure. a. The unit cell. b. The calcium rich layer of the (-101) face. c. A layer parallel to the (101) face.

Figure 4. Histogram showing the average number of COM crystals (all formations) observed under each phosphatidylglycerol monolayer, held at the conditions indicated. The subphase is RS ) 5 calcium oxalate at 25 °C and pH 7. Samples were removed from the LB trough after 4 h, and the reported surface densities, determined from SEM, are averages over nearly 40 mm2. The series of experiments at the same surface pressure, 20 mN/m, varies the headgroup packing while maintaining similar monolayer fluidity. The constant area series, 95 Å2/molecule, compares monolayers with the same headgroup packing but different film fluidity.

differently at the air/water interface, with the chain length and structure of the lipophilic tails determining the molecular packing and the state of the monolayer. These different properties are revealed as the lipid monolayers are laterally compressed on the Langmuir trough. Plots of the surface pressure vs surface area isotherms for the four lipids are shown in Figure 4. The lipids DMPG, POPG, and DOPG form two-dimensional liquids when compressed, or more precisely, they form liquid expanded (LE) phases with no orientational or positional order of the molecules. On the other hand, DPPG is in the LE state only below a surface pressure of 5 mN/m, above

approximately 80 A2/molecule. At higher pressures, the monolayer begins to condense, forming a pure liquid condensed (LC) phase at areas smaller than 45 A2/molecule, a phase in which the molecules do orient with short-range positional order. With all four isotherms on the same plot, Figure 4 shows how two sets of experiments can be envisioned.41 By holding the monolayers at a common pressure, in this case 20 mN/m, the headgroup spacing is changed while maintaining roughly similar compressibility. In the other set of experiments, the area per molecule is held constant, 95 A2/molecule, but each monolayer experiences a different surface pressure and different compressibility.

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Experiments were performed at the pressures and areas indicated in Figure 4 and crystal formation was analyzed with SEM. For the same pressure/different area series, the general trend is that more crystals are seen at the monolayers with smaller corresponding mean molecular area (mma) per molecule. It should be remembered that the DPPG monolayer is in the LC phase at this pressure, whereas the other three are in the LE phase, although the trend still applies if only the three LE monolayers are considered. This experiment suggests that the higher charge density at the monolayer leads to increased COM precipitation. It is interesting that the selectivity for the (1h01) face was similar for each monolayer, even though the organization of the molecules within the monolayer is different. This observation argues against the importance of an epitaxial match between a preformed monolayer template and the incipient crystal face. In the same area/different pressure experiment, the trend is that more crystals are formed at lower pressure. Each monolayer is in the LE phase, and the charge density at the interface is similar, but the mobility of the phospholipid molecules within the monolayer is greater at lower pressure. The data in Figure 4 can also be used to compare each lipid under two different sets of conditions. Except for the DOPG film, at which no crystals were observed, there is an increase in crystal formation as the monolayers are expanded. The result is not unique to the glycerol headgroup, as the same trend was observed with the choline lipid, DPPC. Expansion leads to a decrease in the average charge density at the monolayer and an increase in the mobility of the phospholipid molecules. The results show that the highest density of crystals form under the phospholipid monolayer that has the potential to achieve the smallest mma. The monolayer does not need to be held in a compressed state, but if it is capable of organizing in a tighter packing arrangement, then crystal precipitation is increased. The monolayer does not need to be preorganized, but crystal formation increases if the monolayer can sufficiently concentrate charge and reorganize to stabilize the growing crystal surface. Our experiments on calcium oxalate support observations on other crystal systems. Studying aspartic acid under amino acid monolayers, Cooper et al.42,43 demonstrated enhanced nucleation rates at medium and low pressures. The authors proposed that the greater compressional freedom of films at low pressure allows mismatches between the film and the nucleating crystal face to be accommodated. Similarly, highly organized films are not necessary to promote calcium carbonate precipitation. The early studies by Mann and Heywood reported increased precipitation under LE phases relative to LC monolayers,44 and Ahn and coworkers45 provided evidence from in situ FTIR that the monolayer reorganizes upon calcite precipitation. It appears to be general that the compressibility of the monolayer and the potential of the monolayer to achieve an optimum area per molecule are more important characteristics that lead to heterogeneous crystallization than is providing a prearranged template. So what is the role of the organic interface and how does it interact with the COM crystal? There is no apparent geometric match between the organized phospholipid monolayers and the different COM crystal faces.32,33 Also, the fact that crystals form with the same crystal face selectivity at monolayers with very different intermolecular spacing41 strongly suggests that a templating effect does not operate. Similar conclusions have (42) Cooper, S. J.; Sessions, R. B.; Lubetkin, S. D. Langmuir 1997, 13, 7165. (43) Cooper, S. J.; Sessions, R. B.; Lubetkin, S. D. J. Am. Chem. Soc. 1998, 120, 2090. (44) Mann, S.; Heywood, B. R.; Rajam, S.; Walker, J. B. A. J. Phys. D Appl. Phys. 1991, 24, 154. (45) Ahn, D. J.; Berman, A.; Charych, D. J. Phys. Chem. 1996, 100, 12455.

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Figure 5. Crystals appear as bright spots at the LE/LC phase boundary of a DPPC monolayer compressed to 5 mN/m over an RS ) 5 calcium oxalate subphase at 21.8 °C after 16 h. The dark background is the LE phase, and the light gray is the LC phase. The scale bar represents 100 µm.

been reached in related experiments on other mineral systems.46 However, there is no doubt that the lipids catalyze precipitation and that (1h01) is stabilized by the lipids. The high surface density of Ca2+ on this face and low coordination number makes phospholipid binding favorable, greatly lowering the surface energy. The lipid:calcium ion ratio will be less than one because the area per calcium ion on the surface is smaller than the cross sectional area of the lipid. Nevertheless, the binding energy is sufficiently favorable that the system will try to bind as many lipids as will fit, which is why the lipids that can achieve closer packing are more active. The same interactions that favor (1h01) selectivity should also come into play during the nucleation event and can help explain why the monolayer catalyzes precipitation. The lipid-surface interaction lowers the surface energy, greatly stabilizing crystal nuclei and lowering the activation barrier to precipitation.

Role of Phase Boundaries If the mobility of molecules within the monolayer can influence crystallization, the effect of phase boundaries becomes an important question. Indeed, preliminary experiments performed in our labs41 and by other groups47 on mixed-phase systems showed evidence of crystals forming at phase boundaries. To explore the role of phase boundaries in more detail, we undertook studies of COM precipitation at two kinds of mixed-phase monolayers.31 The first was a single phospholipid in equilibrium at a phase change, either at gas/liquid phase (G/LE) coexistence or LE/LC coexistence. The second set of experiments involved a phase separated binary mixture of different lipids. Figure 5 shows a BAM image of calcium oxalate crystals precipitating at a DPPC monolayer held at LE/LC coexistence over a supersaturated subphase.31 A significant number of crystals appear as bright spots at the boundary between the LE phase (dark background) and the LC phase (light gray). A phase (46) DiMasi, E.; Olszta, M. J.; Patel, V. M.; Gower, L. B. CrystEngComm 2003, 5, 346. (47) Schief, W. R.; Dennis, S. R.; Frey, W.; Vogel, V. Colloids Surf. A 2000, 171, 75.

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Figure 7. Schematic of a lipid raft monolayer model with the sphingomyelin and cholesterol rich liquid ordered phase segregated from a liquid disordered phospholipid phase.

Figure 6. BAM images of a 1:1 DPPC/DMPC monolayer held over an RS ) 5 subphase at 21.8 C after 5 h. The dark gray background is LE phase containing DMPC, the light gray islands are DPPC in the LC phase, and the bright spots are COM.31 At 25 mN/m and the two lipids are phase segregated. The arrows point at crystals precipitating on the DPPC domains. At lower pressure, 18 mN/m, the LE matrix now consists of both DMPC and DPPC. COM grows both at phase boundaries and at LC domains.

boundary can also be created by mixing two lipids that phase segregate. For this, we chose a 1:1 mixture of DMPC and DPPC. Under our experimental conditions, only DPPC can form an LC phase when compressed. At low surface pressure, the lipids mix in an LE phase, but as the monolayer is compressed above 15 mN/m, the DPPC begins to segregate and form condensed domains surrounded by the remaining DPPC/DMPC LE mixture (Figure 6). As the pressure is increased further, the LC domains become larger, and by 25 mN/m, the phase segregation appears to be complete with DPPC in the LC phase and DMPC remaining in the LE phase. COM crystals grown at the phase segregated monolayer appear exclusively on the DPPC domains (Figure 6). Even after 24 h, crystals are associated with the DPPC domains and do not appear at the phase boundaries. Even though the monolayers in Figures 5 and 6a are both held at the same temperature and subphase composition, and both have LE/LC phase boundaries, the COM crystals form exclusively at the phase boundary in the single component DPPC monolayer and away from it at the LC phase in the DPPC/DMPC binary mixture. The mixed lipid system gives a different result if the monolayer is held at lower pressure (Figure 6b). At 18 mN/m, the DPPC has no longer completely segregated, so the LE matrix contains both DPPC and DMPC. Under these conditions, COM again precipitates at the LC domains, but crystals are now observed at the phase boundary. Unlike the situation in Figure 6a in which DPPC and DMPC are completely segregated, at the lower pressure, DPPC is in equilibrium between the LC and the mixed DPPC/DMPC LE phase. A difference is that the boundary is now more dynamic and this dynamic exchange between phases appears to be a condition for crystals to form at the phase boundary. It is reasonable to think that a phase boundary provides a favorable site for nucleation. Relief of the line tension associated

with the two-dimensional phase boundary will contribute to the overall free energy gain upon precipitation. There is a high density of molecules at the phase boundaries, and at the same time, a dynamic boundary with molecules exchanging between phases provides the fluidity necessary for the lipid molecules to reorganize to accommodate the nucleating crystal face. The situation is different for the phase segregated binary mixtures. Without molecular exchange between phases, the compliancy of the phase boundary is lost and a kinetic barrier develops that limits the ability of the lipids to reorganize to stabilize the incipient crystal. The above mechanism is plausible, but other possibilities need to be acknowledged. Schief et al.47 have suggested a mechanism whereby dipole/dipole interactions between crystals and the polar lipid assemblies attract crystals formed in the LE phase to the boundaries. These forces are clearly present, although they may be screened in high ionic strength solutions. Nevertheless, the BAM and SEM observations we have described would not be able to detect this process.

Studies at “Lipid Rafts” The term “lipid raft” refers to structures derived from detergent insoluble components of plasma membranes.48 When reconstituted in model systems,49 rafts form as segregated domains, rich in sphingolipids and cholesterol, that are more ordered than the balance of the lipid matrix (Figure 7). Their existence in living cells remains controversial, but lipid rafts are proposed to play roles in many membrane functions, including transmembrane signaling, regulation, and cellular compartmentalization. This recent interest in the concept of lipid rafts draws attention to the fact that phase heterogeneities are indeed present in cellular lipid membranes. We therefore investigated calcium oxalate precipitation in the presence of phase-separated assemblies of lipid raft composition.50 Raft-forming monolayers were prepared from a 2:1:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, sphyngomyelin, and dihydrocholesterol over a calcium oxalate subphase. Compression to 32 mN/m, a pressure comparable to that of membrane bilayers, causes separation of liquid ordered (LO) domains from the surrounding disordered phase (Figure 8a). The term liquid ordered refers to condensed multicomponent domains, similar to LC designation of a single component lipid phase diagram. The rafts appear as light gray islands in the BAM images. Initially 10-20 µm in diameter, the rafts are fluid and merge to form larger domains, up to several hundred µm across. When calcium oxalate precipitates at the lipid raft monolayers, approximately 80% of the crystals are observed at the LO phase boundaries (Figure 8b), consistent with the previous experiments with pure phospholipids monolayers. However, the remaining 20% of crystals appeared to form in the expanded phase, (48) Pike, L. J. Biochem. J. 2004, 378, 281. (49) Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Biophys. J. 2001, 80, 1417. (50) Benitez, I. O.; Talham, D. R. J. Am. Chem. Soc. 2005, 127, 2814.

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Figure 8. BAM images of a 2:1:1 POPC/SM/dihydrocholesterol monolayer at 32 mN/m over an RS 5 subphase at 25 °C after (a) 0 h and (b) 3.5 h. The dark background is POPC, the light gray islands are SM/dihydrocholesterol, and the bright spots are COM. The arrows point at crystals precipitating at the phase boundaries. The scale bars represent 100 µm.

something we had not observed in the earlier single-component and two-component studies. Brewster angle microscopy is powerful as an in situ measurement, but its spatial resolution is limited, so submicron features in the monolayer structure are missed. Atomic force microscopy can provide this detail. An AFM image of a domain edge from a 2:1:1 POPC/sphingomyelin/dihydrocholestrol monolayer that has been transferred onto mica is shown in Figure 2d. In addition to the large LO domain, submicron rafts are also present. Images of monolayers transferred from a few minutes to hours after spreading all show these small features, suggesting they are present at all times. It is likely then that the calcium oxalate crystals that appear by BAM to form in the LE phase are actually precipitating at these small LO rafts.

Conclusions Urinary stone formation, although pathological, includes mechanistic similarities with purposefully directed biomineralization. Concepts regarding the role of the organic matrix during biomineralization can be considered when investigating COM formation in urinary stones. At the same time, calcium oxalate provides a useful platform for studying general questions related to biomineralization. Langmuir monolayer studies continue to prove useful for elucidating the mechanisms of heterogeneous

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nucleation of inorganic particles at organic interfaces. With respect to calcium oxalate, the work reviewed here demonstrates that lipid interfaces catalyze COM precipitation, and Langmuir monolayers can be used to learn about possible mechanisms, permitting systematic studies on the membrane features that influence crystallization. Among our conclusions to date is that the lipid interface does not need to be preorganized to facilitate crystallization, but it should be able to achieve the packing necessary to sufficiently concentrate charge and contribute to surface stabilization through adsorption. The interface is more effective if it has the potential to form an arrangement that favors the incipient crystal face. At the same time, increased molecular mobility and film compressibility lead to enhanced crystal precipitation, implying that interactions between the organic layer and the inorganic crystal face are cooperative. A rigid monolayer might be able to template a crystal face, but if the lattice match is not perfect, it may actually suppress nucleation and growth. A dynamic monolayer interface can reorganize to achieve an arrangement that is favorable for nucleation. This observation parallels conclusions drawn from cell culture studies.11 Phase boundaries, therefore, become favorable sites for crystallization. Relief of the energy associated with phase boundary contributes to the overall energy gain upon inorganic precipitation. Phospholipid molecules at boundary sites are the most likely to rearrange to accommodate an incipient crystal. However, a static phase boundary can produce a kinetic barrier to crystallization. The observations support the idea that membrane heterogeneities can act to catalyze calcium oxalate precipitation and may help explain observations from tissue culture studies that revealed increased calcium oxalate crystal growth and crystal attachment at dynamic regions at the lipid interface. For example, crystal attachment to cells isolated from the inner medullary collecting duct has been correlated with membrane fluidity.11 Membrane damage, which is prevalent after exposure to oxalate or calcium oxalate crystals may lead to exceptionally fluid sites that can catalyze crystal nucleation or adhesion. Furthermore, millions of tubular epithelial cells are discarded daily into the urine.51 It is highly likely that membraneous material from these cells becomes incorporated into growing stones, either by inducing nucleation or by adsorption to growing crystals. There are still many, many questions involving the role of lipids in urinary stones. The role of phase boundaries needs more study. In addition to nucleation, the adsorption of lipids to crystal surfaces and their role in crystal aggregation are important unresolved questions. Related is the issue of crystal attachment to sites within the urinary tract, which could also involve lipid interfaces. Model membrane systems such as Langmuir monolayers can be used to investigate basic crystal/lipid interfacial phenomena associated with each of these processes. Acknowledgment. We thank the National Institutes of Health, Grant RO1 DK59765, for support of this work. LA052503U (51) Prescott, L. F. Clin Sci 1966, 31, 425.