Evidence That Brushite Is the Nidus of Renal Stones Originating As Calcium Phosphate (Nephrolithiasis/Acid Urine/X-Ray Diffraction)

Proc. Nat. Acad. Si. USA Vol. 68, No. 7, pp. 1456-1460, July 1971

Spontaneous Precipitation of Brushite in Urne: Evidence that Brushite Is the Nidus of Renal Stones Originating as Calcium Phosphate (nephrolithiasis/acid urine/x-ray diffraction)

CHARLES Y. C. PAK*, EDWARD D. EANESt, AND BELLE RUSKIN* * Section on Mineral Metabolism, Endocrinology Branch, National Heart and Lung Institute, Bethesda, Maryland 20014; and t Laboratory of Biological Structure, National Institute of Dental Research, Bethesda, Maryland 20014 Communicated by Robert W. Berliner, April 26, 1971

ABSTRACT Further evidence that brushite plays a cium, or, in patients with hyperparathyroidism, by its surgical regulatory role in renal stone formation was provided by the identification of brushite as the first precipitate that correction. In the second group, the urine is supersaturated appears in supersaturated urine by spontaneous precipita- primarily because of an abnormally high concentration of tion. Calcium chloride was added to induce supersatura- HP042- ions; stone formation is prevented when the urinary tion in urine specimens from twelve subjects with and concentration of HP042- (and the activity product for twelve subjects without nephrolithiasis. brushite) is lowered avoidance of alkali or The first precipitate in all specimens with pH below 6.9 by by elimination of was identified as brushite by x-ray diffraction and shown infection. to have a calcium-phosphorus ratio of approximately 1.0. The conclusion that brushite is indeed the initial nidus that The activity product of [Ca'+i X [HP0421- necessary to forms from urine onto an organic matrix was supported by the produce a precipitate ranged from 2.2 to 3.5 times the demonstration that the activity product for brushite controls solubility product of brushite, but the range and mean were the same for both groups of subjects. nidation on an artificial "matrix" composed of collagen (4). The activity product of [Cal+] X [HPO42-] in the super- The present studies were undertaken to identify the initial natant (after spontaneous precipitation) was not signifi- precipitate when crystals form de novo in urine by spontaneous cantly different from that obtained after incubation of the precipitation (2, 5, 6). The results indicate that this precipi- same urine specimen with synthetic brushite. tate is also brushite, but that it does not appear until the These results provide conclusive evidence that brushite constitutes the solid phase formed by spontaneous pre- activity product of [Ca2+] X [HPO42-] in the urine exceeds cipitation from acidic urine supersaturated with respect two-times saturation. to calcium and phosphorus; they suggest that the nidus For these studies, a formation product ratio is defined. This for calcium-containing renal stones is brushite as well. is the ratio of the activity product of [Ca2+] X [HP042-1 Physicochemical factors controlling the formation of calcium- necessary to form a precipitate from a given urine specimen containing renal stones cannot be understood until the initial (with or without added Ca2+) to that of another aliquot of the precipitate is identified. When this precipitate forms on an same urine specimen after equilibration with brushite. This organic matrix or is added to a preexisting stone, it is com- ratio allows quantitative calculation of the tendency for pre- posed of brushite (CaHPO4 * 2H20), and is deposited when the cipitation de novo. activity product of [Ca2+] X [HP042-] in the urine exceeds MATERIALS AND METHODS the activity product (K.,) of brushite (1-4). Clinical data In earlier studies, a ratio of the solubility product of the Twelve patients with a history of recurrent passage of calcium- actual urinary K.,, for brushite to that of urine with an added containing renal calculi (nephrolithiasis group), and twelve excess of solid brushite allowed quantitative calculation of the subjects without stones (control group) were evaluated tendency for stone growth and corrected for any error in the (Table 1). In the nephrolithiasis group, six patients had calculation of activities for Ca2+ and HP042- (2). idiopathic hypercalciuria (7), six had normal urinary calcium. These results were used to explain growth of stones in All had passed several renal calculi some containing calcium. patients with hypercalciuria and some with urine Seven were men and five were women; the mean age was 40 persistently alkaline as a result of therapy with alkali or of years. The control group consisted of normal volunteers, andof urinary-tract infection by urea splitters. In the first group, the patients with essential hypertension, vitamin D-resistant urine is supersaturated with respect to brushite, primarily rickets, postoperative hypoparathyroidism, or idiopathic of because an abnormally high concentration of Ca2+ ions. osteoporosis. Three were men and nine were women; the mean Stone formation is prevented when the concentration of age was 28 years. All patients received a diet containing 400- urinary calcium (and thus the activity product for brushite) 600 mg/day of calcium for 5 or more days before, and during, is lowered by the feeding of nonabsorbable chelators of cal- the study. One 24-hr urine specimen was obtained from each patient; it was kept cold without preservative (1, 2). The * Requests for reprints should be addressed to Dr. Charles Y. C. urinary calcium concentration among the patients with Pak, 8N212, National Heart and Lung Institute, Bethesda, Md. hypercalciuria was over 200 mg/day, with a mean value of 20014. 269 mg/day; in the remaining subjects and controls it aver- 1456 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Precipitation of Brushite in Urine 1457 TABLE 1. Urinary composition Nephrolithiasis Nephrolithiasis group group (hypercalciuria + Control Hypercalciuria Normocalciuria normocalciuria) group No. of Patients 6 6 12 12 Ca, mg/day 269 i 69 130± 43 200± 90 130± 92 Mg, mg/day 99 ± 44 76 ± 37 87 ±- 41 94 ± 41 P, mg/day 893 4± 314 793 i 205 843 ± 258 640 ± 125 Na, meq/day 127 ± 56 85 ± 44 106 + 53 72 ± 44 K, meq/day 44 ± 18 51 ± 15 48 ± 16 54 ± 13 pH 5.83 ±t .29 5.96 ± .38 5.89 ± .33 6.19 ± .37 Total volume, ml/day 2378 i 578 2187 i 575 2282 i 559 2024 i 599 Values are given as mean ±SD. aged 130 mg/day (Table 1). There were no significant differ- activity product was lower than the highest product obtained. ences between groups in the mean total urine volume, or pH, The curves for precipitation were biphasic (Fig. 1), charac- or in the urinary Mg, P, Na, or K concentrations. The initial terized by a region of rapid 'fall' where the activity product pH of all 24 specimens was less than 6.7. decreased rapidly during a small increase in the initial activity product, and a region of 'steady-state', where the final Estimation of the degree of saturation of urine with respect to brushite activity product did not change significantly despite a further The activity product of [Ca2+] X [HP042-] (K5p) of urine was increase of the initial activity product. determined as described elsewhere (2). The degree of satura- The region of rapid fall was extrapolated to the curve of no tion of urine with respect to brushite was calculated as precipitation; the activity product at the junction (Fig. 1, follows. Kp of urine was calculated before and after incuba- arrow) represents the formation product (5). tion with brushite (CaHPO4.2H20, Mallinkrodt). The ratio Calculation of the formation product ratio of the activity product of urine before incubation (Kp i) to The final activity product after 1 day of incubation of urine that of urine supernatant after incubation (Kpf) (activity was than that after 2 or 4 of incubation product ratio) represents the degree of saturation of urine. A specimens higher days value of K.,,i/K~pf of 1 indicates saturation, a value above 1, supersaturation, and a value below 1, undersaturation. I O '/I* All studies were performed in new test tubes without E l I.* visible scratches. Selected experiments were also performed ~ I' with siliconized test tubes; identical formation product ratios 3- Ks were obtained. FINAL II~~~~~~~~~~ 4 Spontaneous precipitation a a | .0do After cellular debris was removed from the urine by centrifu- Ca* FIP04= gation, twelve 10-ml aliquots from each sample were pipetted xCa*~~~~~~~~~IO7*day04 into 20-ml test tubes. 20 mg of brushite was added to tube 1. 0 Tube 2 represented the control, which contained the unaltered 9 urine specimen. Into tubes 3-12, a solution of CaCl2 (30 mg of 5210 15 " 4 20day 2 calcium/ml) was added in progressively larger amounts. 3 drops of chloroform were added to each tube to prevent bacterial contamination. The specimens were incubated at 5 10 15 20 25 30 37°C for 48 hr with constant stirring. The pH of each specimen was adjusted to the original pH with 0.1 N HCl or 0.1 N INITIAL aa ,a X 107 NaOH after 0.5 hr, 6 hr, 24 hr, and 30 hr of incubation. In two specimens, the unaltered urine contained a precipitate after FIG. 1. Calculation of formation product ratio of brushite. The incubation. These two specimens were first diluted with activity product of [Ca2+J X [HP042-] in a urine sample was different amounts of water until precipitation failed to occur varied from its original value of 2 X 10-7 to 2.62 X 10-6 by the after incubation, and then were treated as described. addition of CaCl2, while the pH was maintained at its original After 48 hr of incubation, the specimens were centrifuged at value of 6.50. After 1, 2, and 4 days of incubation, the activity 1400 X g. The activity product of Ca2+ and HP042- of the product of the supernatantwas determined (final activityproduct). supernatant solutions was then calculated: the final, rather When precipitation did not occur, the initial activity product was than the original, pH of urine was used. The activityproduct of equal to the final activity product (no precipitation). When precipitate formed, the final activity product was lower than the the supernatant solution was then compared with that of the initial activity product. The arrow indicates the formation same specimen before incubation (Fig. 1). When there was no product, the lowest activity product at which precipitation com- precipitation, the final activity product was equal to the mences. K8p (shown by dashed horizontal line) is the activity initial activity product (represented by the curve of no product of Ca2+ and HP042- after incubation of urine with precipitation, Fig. 1). When there was precipitation, the final synthetic brushite. Downloaded by guest on September 29, 2021 1458 Medical Sciences: Pak et al. Proc. Nat. Acad. Sci. USA 68 (1971)

4 TABLE 2. Identification of the solid phase by x-ray diffraction

0Ok 0 pH No. of samples Predominant phase FORMATION 3 0 * 0 5.2-6.65 24 brushite 0 0 0 PRODUCT 0 6.90 1 brushite 2 * ° 0 0 RATIO 6.95 1 hydroxyapatite 7.56 1 hydroxyapatite 7.75 1 hydroxyapatite * Nephrolithiasis o Control The pH represents the final pH of the urine sample. In the four 0 x Artif cal sol'n specimens with pH of 6.90 or greater, the initial pH was pur- 5.0 6.0 pH 7.0 posely increased. FIG. 2. The formation product ratio (ratio of formation product and Kp) of brushite in 24 urine specimens. The ratio urine specimens to which no calcium had been added.) It was ranged from 2.12 to 3.50. There was no significant difference packed into a thin-walled glass capillary, 0.5 mm in diameter, between values for the nephrolithiasis group [2.69 i 0.46 (SD)] and examined by x-ray diffraction. The x-ray diffraction from and those for the control group [2.63 + 0.43 (SD)]. N-filtered Cu radiation (X = 1.54 X) diagram was recorded with a 114.6 mm Debye-Scherrer powder camera. Exposure (Fig. 1). Moreover, the activity product of the urine specimen time was 3 hr. The diffraction diagram was identified by visual after its incubation with synthetic brushite (K8., dashed line, comparison with reference patterns. Fig. 1) was equivalent to that after spontaneous precipitation Chemical composition of the solid phase had proceeded for 2 or 4 days of incubation. Thus, it requires at least 2 days for the newly-formed brushite to reach a steady The precipitates obtained from 11 urine specimens were dis- state with the ambient solution. solved in 1.2 N HCl and analyzed for calcium and phosphorus. The solubility of synthetic brushite in urine (K8p) was, The final pH of urine ranged from 5.12 to 7.75. therefore, employed for the calculation of the formation Calculation of the formation product ratio in an product ratio, the ratio of the formation product to the K8p. artificial solution It is an estimate of the number of times the urine specimens must be supersaturated with respect to brushite for spon- By the same technique described for urine, the formation taneous precipitation to occur. product ratio was calculated for an artificial solution at pH 6 For one selected urine specimen, the formation product ratio that contained 100 mnI Na, 20 mM K, and 10 mM of P. was calculated repeatedly after the urinary pH had been RESULTS changed from 6.0 to 6.8. The formation product ratio was found to be independent of pH. The formation product ratio The formation product ratio was greater than 2 in all 24 urine Identification of the solid phase by x-ray diffraction specimens tested-thus, all specimens were at least twice For each urine specimen, the precipitate from one of the tubes supersaturated with respect to brushite before spontaneous (usually the second tube to show precipitation) was separated precipitation occurred (Fig. 2). There was no significant by centrifugation, washed in methanol, and dried in air at difference in the formation product ratio between specimens 100C. (Two of the precipitates had formed from unaltered from the nephrolithiasis group and those from the control group; the mean formation product ratio in the nephrolithiasis

- 2-0 group was 2.69 ± 0.46 (SD), that in the control group was * Nephrolithios's o Control 2.63 ±t 0.43. Within the nephrolithiasis group, those with 0 hypercalciuria had a slightly lower mean formation product than those with normal urinary calcium, but the difference DECREASE IN +20 0. - was not significant (2.54 ± 0.31 as compared to 2.84 i 0.46). URI N ARY The formation product ratio of brushite in the artificial solu- Ca +40 tion was 2.18 ± 0.03 (SD). This value is close to the value of FROM o 0o 2.3 reported by Hlabse and Walton (8). PRECIPITATION + 6 0 00 * 0 Dependence of spontaneous precipitation on the degree of to + 80 saturation of urine with respect brushite (Fig. 3) Four tubes were selected from each study of spontaneous + 100 0 2 3 4 5 6 7 E precipitation: the original urine specimen without added ACTIVITY PRODUCT RATIO calcium, the specimen with the highest calcium concentration that did not show precipitation, and the supernatant fluid FIG. 3. Dependence of spontaneous precipitation on the degree from the first two specimens that did show precipitation. From of saturation of urine with respect to brushite. Precipitation was the supernatant fluid, the activity product ratio and the accompanied by a decrease in calcium concentration in the speci- change in the concentration of calcium that had resulted from men of more than 5%. Precipitation did not occur from speci- were was always mens that were undersaturated with respect to brushite (activity precipitation calculated. Precipitation product ratio of less than 1), or even from specimens that were accompanied by a decrease in the calcium concentration of the up to twice supersaturated. supernatant of more than 5%. Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Precipitation of Brushite in Urine 1459

Precipitation did not occur from specimens that had an 2.0 - activity product ratio of less than 2 (Fig. 3); precipitation tended to be greater the more the original specimen was super- 0 0 saturated. Qualitatively similar dependence of spontaneous 1. 5 precipitation on the activity product ratio was shown for urine specimens from the nephrolithiasis group and those from Co/P 1.0 0 the control group. Identification of the solid phase: solubility studies 0.5 The activity product of the supernatant from urine specimens, 2 days after spontaneous precipitation had occurred, was essentially the same as that after incubation with brushite (4.5 X 10-7 ± 0.9 X 10-7 (SD) as compared to 4.9 X 10-7 i 5 6 7 8 1.2 X 10-7); this suggests that the solid phase formed by pH spontaneous precipitation is predominantly brushite. FIG. 4. Ca/P of precipitates. The solid phase collected from Identification of the solid phase: x-ray diffraction studies urine with a final pH of 6.9 or less had Ca/P of about 1 (closed All 24 preparations of the solid phase formed by spontaneous circles). Above pH 6.9, Ca/P was 1.5 (open circles). precipitation from the urine of the 24 subjects were predominantly brushite as determined by x-ray diffraction (Table 2). The present study provides conclusive evidence from solu- All were formed from urine with a pH near that of the original bility characteristics, chemical composition, and x-ray diffrac- urine (range, 5.2-6.65). No trace of calcium oxalate, octa- tion that the solid phase formed by spontaneous precipitation calcium phosphate, or hydroxyapatite was identified. from urine in the absence of organic matrix is also brushite. When a nidus of brushite is exposed to an alkaline medium, it Identification of the solid phase: is hydrolyzed into another phase of calcium phosphate, such molar calcium: phosphorus ratio (Ca/P) as hydroxyapatite (Figs. 4 and 5). These findings-the demon- When the supernatant fluid was at pH 6.9 or less, the Ca/P stration of brushite as the only significant solid phase formed of the solid phase was about 1, which suggests that the solid either under the influence of an organic matrix or by spon- phase was brushite (Fig. 4). All these specimens were shown to taneous precipitation, and the evidence that brushite can be be brushite by x-ray diffraction. transformed into other calcium phosphates -support the con- Effect of pH and aging on crystal structure clusion (2) that at the normal acid pH of urine, brushite Four preparations of the solid phase were collected from urine probably constitutes the crystal nidus and may serve as a specimens whose pH had been titrated to greater than 6.65 precursor for renal stones not only of brushite but also of with 0.1 N NaOH. The specimens collected at a final pH of other calcium phosphates. 6.95, 7.56, 7.75 were hydroxyapatite as determined by x-ray At more alkaline pH, the stable phase of calcium phosphate diffraction (Table 2); in two of these precipitates the Ca/P is hydroxyapatite (5) (Figs. 4 and 5). Thus, hydroxyapatite, ratio was 1.5, a value consistent with that of calcium-deficient rather than brushite, might regulate the formation of renal apatite (Fig. 4). stones from alkaline urine. However, the possibility that For one urine specimen in which the pH was kept between brushite is formed before hydroxyapatite cannot be excluded 6.3 and 6.5, the solid phase was collected at various times even under these conditions; the initial calcium phosphate after the onset of spontaneous precipitation. All precipitates precipitated at alkaline pH has a Ca/P ratio of about 1, the obtained between 0.5 hr and 21 days contained predominantly ratio for brushite (9). brushite, as determined by x-ray diffraction. In another experiment (Fig. 5), the solid phase was collected * pH 6.0 at 0.5 hr, and again daily for 5 days after the onset of spon- o pH 7.4 taneous precipitation, while the urinary pH was kept near 6.0. All precipitates were predominantly brushite, as estab- lished by x-ray diffraction. The activity product of [Ca2+] X [HP042-] of the supernatant fluid decreased during the first xIO 7 2 days, but did not significantly change further during the subsequent 3 days. After two days of incubation at pH 6.0, a portion of the supernatant fluid was withdrawn and its pH was adjusted to 7.4 with 1 N NaOH. The activity product of [Ca2+] X [HP042-] of this specimen decreased markedly, and the solid phase that precipitated proved on x-ray diffrac- 0 2 3 4 5 tion to be hydroxyapatite rather than brushite. DAYS DISCUSSION FIG. 5. Effect of pH and aging on the activity product of We Ca2+ and HP042-. At pH 6, the activity product of the urine have previously proposed a new scheme for the patho- supernatant decreased during the first day after spontaneous genesis of renal stones containing calcium phosphate, wherein precipitation, but it did not change significantly after the first brushite plays a regulatory role (1, 2). When calcium salt day. Change of pH to 7.4 markedly lowered the activity product. forms from urine on an artificial matrix of collagen, the initial The solid phase at pH 6 was identified as brushite by x-ray mineral phase formed (or the crystal nidus) is brushite (4). diffraction; that at pH 7.4 was hydroxyapatite. Downloaded by guest on September 29, 2021 1460 Medical Sciences: Pak et al. Proc. Nat. Acad. Sci. USA 68 (1971)

The formation product ratio of most urine specimens was The current study does not discriminate between the pre- above 2.18 (Fig. 2), the figure obtained for the artificial solu- cipitation-crystallization theory (14, 15) and the matrix tion. The difference is probably the result of inhibitors of theory (11, 16, 17) of stone formation. However, it emphasizes nucleation, such as pyrophosphate (10) or peptides (11) in the the validity of the model proposed wherein brushite plays a urine. However, inhibitors of nucleation probably do not play regulatory role, irrespective of the mechanism by which a significant role in the pathogenesis of renal stones: the formation of the calcium-containing renal calculus begins. formation product ratios from the nephrolithiasis group were not significantly different from those from the control group We gratefully acknowledge the technical assistance of G. N. (Fig. 2). If the urine specimens in the nephrolithiasis group Martin. as has been proposed by some lacked inhibitors of nucleation, 1. Pak, C. Y. C., E. C. Diller, G. W. Smith II, and E. S. Howe, workers (11, 12), the formation product ratios from these Proc. Soc. Exp. Biol. Med., 130, 753 (1969). urine specimens should have been lower than those from the 2. Pak, C. Y. C., J. Clin. Invest., 48, 1914 (1969). control group. A similar conclusion was reached by Robertson 3. Pak, C. Y. C., J. W. Cox, E. Powell, and F. C. Bartter, and colleagues (13), and by us, previously, from studies of the Amer. J. Med., 50, 67 (1971). 4. Pak, C. Y. C., and B. Ruskin, J. Clin. Invest., 49, 2353 calcification of collagen (4). (1970). We believe that the important determinant in" the patho- 5. Strates, B. S., W. F. Neuman, and G. J. Levinskas, J. genesis of renal stones containing calcium phosphate is the Phys. Chem., 61, 279 (1957). state of saturation of urine with respect to brushite. To allow 6. Robertson, W. G., M. Peacock, and B. E. C. Nordin, Clin. Sci., 34, 579 (1968). the formation de novo of a precipitate of brushite, the activity 7. Henneman, P. H., P. H. Benedict, A. P. Forbes, and H. R. product of Ca2+ and HP042- of urine must be greater than the Dudley, N. Engl. J. Med., 259, 802 (1958). formation product. In the presence of an organic matrix, the 8. Hlabse, T., and A. G. Walton, Anal. Chim. Ada, 33, 373 urine may be only slightly supersaturated for the nidus to (1965). form, since the formation product under these circumstances 9. Neuman, W. F., T. Y. Toribara, and B. J. Mulryan, Arch. Biochem. Biophys., 98, 384 (1962). is only slightly greater than the activity product (4). How- 10. Fleisch, H., and S. Bisaz, Amzer. J. Physiol., 203, 671 (1962). ever, for a nidus of brushite to form spontaneously, the urine 11. Howard, J. E., W. C. Thomas, L. M. Barker, L. H. Smith, must be 2-3.5 times supersaturated with respect to brushite and C. L. Wadkins, Johns Hopkins Med. J., 120, 119 (1967). (Figs. 2 and 3). Many patients with idiopathic hypercalciuria 12. Thomas, W. C., and J. E. Howard, Trans. Ass. Amer. urine whose Physicians, 72, 181 (1959). and nephrolithiasis excrete specimens activity 13. Robertson, W. G., J. Hambleton, and A. Hodgkinson, product ratios exceed the range of formation product ratios Clin. Chim. Acta, 25, 247 (1969). reported here (1). Further, normocalciuri6 patients with 14. Vermuelen, C. W., E. S. Lyon, and W. B. Gill, Invest. Urol., stones may excrete urine with a similar degree of super- 1, 370 (1964). saturation during infections of the urinary tract with urea- 15. Vermuelen, C. W., E. S. Lyon, and F. A. Fried, J. Urol., 94, 176 (1965). splitting organisms. Thus, most patients with nephrolithiasis 16. Boyce, W. H., and J. S. King, Jr., Ann. N.Y. Acad. Sci., could form a nidus of brushite either by spontaneous precipi- 104, 563 (1963). tation or by nucleation of an organic matrix. 17. Boyce, W. H., Amer. J. Med., 45, 673 (1968). Downloaded by guest on September 29, 2021