May, 1959
EFFECT OF MOLAR CA/P ON CRYSTALLIZATION OF BRUSHITE AND APATITE
725
THE EFFECT OF THE MOLAR Ca,/P RATIO UPON THE CRYSTALLIZATION OF BRUSHITE AND APATITE1 BY JAMES S. ELLIOT,ROBERT F. SHARPAND LEONLEWIS From the Poliomyelitis Respiratory and Rehabilitation Center, Faimnont Hospital, S u n Leandro California; The Division of Urology, Department of Surgery, University of Culifornia School of Medicine, S a n Francisco, &ulifornia; The Department of Medicine, Stanford University School of Medicine, S u n Francisco, California Receiued October SO, 1068
Data in the literature indicate that calcium phosphate precipitates from saturated solution as brushite up to pH 6.2 and as apatite at higher pH levels. In the course of a laboratory investigation concerning the chemical factors responsible for renal calculus growth in para1 zed persons, precipitates in equilibrium with urine saturated with calcium phosphate and having an average molar Ca/$ ratio of 3/20 were found to consist of brushite up to pH 6.61 and of apatite a t higher p H levels. I n urine specimens with very low Ca/P ratios due to the addition of sodium phosphate the precipitates were composed of brushite up to pH 6.93. These observations suggested that the Ca/P ratio in parent solutions and in urine might have considerable effect on the relationship between equilibrium pH and the composition of the solid phase. In order to test this hypothesis, solutions of calcium phosphate were prepared having molar Ca/P ratios ranging from 3/1 to 1/100, with the ionic strength maintained a t 0.32 by the addition of sodium chloride. Saturation was achieved by adding varying amounts of sodium hydroxide. Solutions were equilibrated with the precipitates for one week at 38”)the solutions filtered, p H of the solution determined and the solid phase examined under polarized light and by X-ray diffraction analysis. Results of this study indicate that the maximum p H a t which brushite is stable is a function of the initial molar Ca/P ratio in solution and the “conversion pH” of brushite to apatite increases with a decreasing Ca/P ratio.
Introduction This paper reports a study of the effect of varying initial C a , P ratios upon the relationship between equilibrium pH and the composition of the solid phase precipitated from solutions saturated with calcium phosphate. The work was undertaken as part of a comprehensive investigation of the urinary chemistry involved in the formation of renal phosphatic calculi, a common and serious problem in severely paralyzed persons. Since calcium phosphate is the principal inorganic constituent of human bone, teeth and dental calculus where it occurs as the mineral, apatite, a number of investigators have studied its solubility in aqueous solution and in serum. Although calcium phosphate is the sole or principal constituent of many renal calculi, little attention has been paid to the factors affecting the crystallization of calcium phosphate from urine. I n common with other biological phosphatic structures, calcium phosphate occurs usually in renal calculi as the mineral apatite. However, in a small percentage of cases it also occurs as the mineral brushite. It is well known that the crystalline structure of calcium phosphate is related to pH of the solution, and it is generally considered that brushite is unstable above pH 6.2, converting to apatite a t higher p H levels. However, in a previous communication,3 i t was shown that in urine saturated with calcium phosphate by the addition of sodium hydroxide, and in urine specimens which were saturated with calcium phosphate without alteration, the precipitates were composed of.pure brushite up t o a p H of 6.61 and pure apatite above this pH level. However, in urine specimens saturated with calcium phosphate by the addition of sodium phosphate, the precipitates consisted of crystalline brushite up to pH 6.93. The average Ca/P ratio (1) Aided by a grant from the National Foundation for Infantile Paralysis, Inc. (2) I n this paper, the “Ca/P ratio” refers t o the initial molar Ca/P ratio in solution. (3) J. S. Elliot, W. L. Quaide, R. F. Sharp and L. Lewis, J . U r d . , 80, 269 (1958).
in urine is 3/20, whereas in the specimens to which sodium phosphate was added, the Ca/P ratios were considerably smaller. The specimen in equilibrium with brushite a t pH 6.93 had a Ca/P ratio of 3/ 1000. These observations suggested that the initial Ca/P ratio in the parent solutions might have considerable effect upon the crystalline structure of calcium phosphate precipitated from aqueous solution and urine. Although Neuman and Neuman4 have stated that the initial Ca/P ratio in solution is a factor which affects the crystalline structure of the precipitate a t equilibrium, a review of the literature has indicated that little attention has been paid to this aspect of the chemistry of calcium phosphate. I n a saturated solution of calcium phosphate both the solubility and the crystalline structure of the precipitate are affected by pH of the solutions. As shown by Hodges who compared the data from a number of investigators, there is a linear relationship between pH of the solution and the molar solubility of calcium. Other observers have shown that pH of the solution affects the structure of the crystalline precipitate. I n 1925 Holt, La Mer and Chown6 and later Dallemagne and Melon,’ and Hodge8 showed that calcium phosphate crystallizes from a saturated solution as secondary calcium phosphate (brushite) below pH 6.2, and as tertiary calcium phosphate (apatite) above pH 6.2 when phosphoric acid is titrated with calcium hydroxide. More recently, by means of precipitation and dissolution experiments, Strates, Neuman and Levinskas9 have shown that brushite is stable a t pH 6.2 (4) W. F. Neuman and M. W. Neuman, Chsm. Reus., 68, 1 (1953). (5) H.C. Hodge, “Some Considerations of the Solubility of Calcium Phosphate,” Conference on Metabolic Interrelations, Transactions of the Third Conference, p. 190,1951. The Josiah Macy, Jr. Foundation. , (6) L. E. Holt, Jr., V. E. La Mer and H. B. Chorvh, J . B i d . d e m . , 64, 509 (1925). (7) M. J. Dallemagne and J. Melon, Bull SOC.Chim. B i d . 28, 566 (1946). ( 8 ) H. C. Hodge. “Some Observations of the Dynamics of Calcification,” Conference on Metabolic Interrelations, Transactions of the Second Conference, p. 73, 1950. The Josiah Macy, Jr., Foundation. (9) B. W. Strates, W. F. Neuman and G . J. Levinskas, THIS JOURNAL, 61, 279 (1957).
JAMESS.ELLIOT, ROBERT F. SHARPAND LEONLEWIS
726
Observations Data relating the initial composition of the solutions, the final pH and the nature of the solid phase are given in Table I. Examination of this ma-
I 7.0 6.6 6.2
TABLE I
5.8 R
RELATIONSHIP BETWEEN COMPOSITION OF THE SOLID PHASE A N D THE FINAL pH AT VARYINQCa/P RATIOS
5.4 5.0
Sample no.
4.6 4.2
Vol. 63
1
1.0 1.5 2.0 2.5 log (Ca/P X 102). Fig. 1.-Relationship between Ca/P ratio and maximum pH at which brushite 1s found as a single crystalline hase. Roman numerals refer to solution series listed in T a b g I. 0
0.5
gri";:
(10) J. Melon and M. J. Dallemagne, Bull. eoc. c h i n . Belu., 66, 180 (1 947). (11) Optical and X-ray diffraction anaIyses were performed by Dr. W. L. Quaide of the Department of Geology, University of Caliornia, Berkeley, California.
(P)9 moles/l. x 10-2
Compn. of solid Brushphase,Apa%
Ca/P
I-1
60.0
2.00
3/1
2 3 11-1 2 8
20.0
2.00
1/1
4
or lower, and a t pH 6.9 it hydrolyzes rapidly to apatite. I n 1947 Melon and Dallemagnelostudied the solid phase a t equilibrium in mixtures of calcium hydroxide and phosphoric acid. In their experiments, brushite did not exist as a stable phase above a pH of 6.0. They observed a linear relationship between initial [PI and final pH in those samples which contained pure brushite. Brushite was found over a pH range from 2.2 to 6.0 with the pH increasing as the initial [PI decreased from 0.270 mole/liter to 2.5 X mole/liter. The molar Ca/P ratio varied from 0.45 to 0.95 and the ionic strength varied over a wide range. It should be noted that the molar Ca/P ratio in all previously reported investigations did not vary greatly from unity. Experimental Seven stock solutions of calcium phosphate were which contained initial molar Ca/P ratios ranging to 1/100. The ionic strength was adjusted to 0.32 (approximating that of normal urine) by added sodium chloride. The initial p H of all solutions was 4.3 and no solid phase was present. Varying amounts of carbonate-free sodium hydroxide were added to 100-ml. aliquots of the stock solution in glass stoppered volumetric flasks. The solutions were preserved with toluene. The flasks were incubated at 38" for one week with daily shaking. The contents of the flasks then were filtered a t 38" under toluene through Whatman #50 filter paper. The pH of each filtrate was determined anaerobically at 38" with the Beckman blood electrode assembly and the Beckman model GS pH meter. Beckman buffer standards of pH 4.03 and 6.97 a t 38" were employed. Precipitates were washed once with 25.0 ml. of distilled water and dried overnight a t 38". Weighing of the dried precipitates indicated that the solid to solution, ratios varied from 5.0 to 70.0 mg. with an average of 40.0 mg. per 100 ml. of solution. The composition of the precipitates was determined by examination under polarized light and by X-ray diffraction analysis.11 Percentage composition of precipitates containing more than one solid phase was estimated by comparison of the X-ray powder patterns with those of known composition. The percentages so determined are considered over all to be accurate to = t l O % . In the case of mixtures containing less than 25% brushite, the percentage error is of the order of & 5 % .
(Ca), moles/l. x 10-3
5 6 111-1 2 3 IV-1 2 3 4 5 v-1 2 3 4 VI-1 2 3 4 VII-1 2 3
6.00
2.00
3/10
6.00
4.00
3/20
3.00
3 .OO
1.50
3.00
6.00
15.0
1/10
1/20
1/100
pH
ite
4.31 4.58 4.78 4.92 5.00 5.01 5.08 5.18 5.31 5.72 5.79 5.83 5.41 5.88 5.91 6.03 6.16 6.22 6.30 6.50 6.61 6.21 6.28 6.41 6.58 6.50 6.74 6.81
100 100 40 100 100 50 35 40
tite
60
50 65 60 100
100 100 100
100 100 35
100 100 10
65 100 100
90 100
100 100 100 100 100 100 100
terial will show that apatite occurred as a solid phase over a pH range of 4.78 to 6.81, and brushite occurred over a pH range of 4.31 to 6.74. Further, it is apparent that the maximum pH a t which brushite is the single crystalline solid increases with a decreasing Ca/P ratio. I n Fig. 1 the value, log ([Ca]/[P] X lo2), has been plotted against the maximum pH a t which pure brushite was found in each of the seven series of solutions. An equation for the regression line was calculated to be: pH = -0.912 log ([Ca]/[P] X lo2) 6.95 with a correlation coefficient of -0.981, indicating essentially a straight line relationship. Summary.-In salt solutions saturated with calcium phosphate having an ionic strength of 0.32, and allowed to equilibrate a t 38" for one week, the precipitates were composed of pure brushite over a pH range of 4.58 t o 6.74. The maximum pH a t which brushite is stable is a function of the initial molar Ca/P ratio in the solution. The "conversion pH" of brushite to apatite increases with a decreasing Ca/P ratio.
+
.
