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Precipitation of calcium carbonates and phosphates. I. Spontaneous precipitation of calcium carbonates and phosphates under physiological conditions
ARCHIVES
OF
BIOCHEMISTRY
Precipitation
AND
BIOPHYSICS
of Calcium
Precipitation
124-138 (1963)
103,
Carbonates
of Calcium
and
Carbonates
Physiological BERNARD From the Department
N. BACHRA, of Biochemistry
OTTO
Phosphates. and
Phosphates
under
Conditions’
R. TRAUTZ
AND
S. LAWRENCE
and Guggenheim Institute for Dental College of Dentistry, New York Received
I. Spontaneous
Research,
SIMON
h’ew York University
June 12, 1963
The slow spontaneous precipitation of calcium carbonates and calcium phosphates was studied under physiological conditions. Calcium carbonate precipitation was prevented by the presence of phosphate ions at concentrations too low for the precipitation of calcium phosphate. The composition of most biological fluids does not allow the deposition of calcium carbonate. The suggestion is made that such deposition will occur under the influence of metabolic processes which locally raise the bicarbonate and lower the phosphate concentration. Aragonite was metastable under the conditions of the experiments. The aragonite present in the early precipitates of calcium carbonate transformed into calcite, when kept in contact with the supernatant solution. The crystallization of apatite is disturbed when bicarbonate is present in the solution. At sufficiently elevated calcium concentrations and bicarbonate/phosphate concentration ratios, an amorphous precipitate of calcium carbonate phosphate formed, which failed to crystallize into apatite when kept in contact with the supernatant solution. The Ca/P molar ratio of the apatitic and amorphous precipitates varied between approximately 2.3 and 1.4. This large variation is due to the coprecipitation of carbonate, HPOa, and Na ions. These and other impurities interfere with the crystallization of the biological apatites and limit the size of the crystallites in bone and dentine. Dental enamel is better crystallized because it contains lesser amounts of these impurities.
tites has been ascribed to the presence of these impurities (la, lb, 2). Crystallized calcium carbonate does not occur in the bone and teeth of the vertebrates (la, 3-5). It is found in this phylum in specific tissues only and no apatite occurs with it. Thus the otoliths in fishes and the statoconia in amphibia contain aragonite (6a, 7-lo), while the statoconia in mammals (6a, 7-9) and the egg shells of birds (lla) and reptiles (Id) contain calcite. The calcified tissues of the invertebrates usually contain calcium carbonate (calcite, aragonite, or vaterite) as the mineral phase. Apatite is rarely found in the invertebrates (6b).
INTRODUCTION
The mineral phase of bone and teeth in the vertebrates gives the X-ray pattern of an apatite. It is a basic calcium phosphate, containing varying amounts of impurities, like carbonate, secondary phosphate, citrate, magnesium, sodium, and potassium. The poor crystallinity of many biological apaI This work was supported in part by a research grant, D-159, from the National Institute of Dental Research, U. S. Public Health Service, and by the Research and Development Command, Office of the Surgeon General Department of the U. S. Army, under Contract No. DA-49.907-MD765. 24
PRECIPITATION
OF CALCIUM
CARBONATES
In some organisms the crystallisat.ion of the mineral phase is disturbed to such a degree that it is completely amorphous. Such amorphous material was demonstrated in small concretions in the tapeworm Taenia taeniaejormis and Cysticewus jasciolaris (lb, lc, 2, 12) and in the carapace and tendon plates of the mantis shrimp and stone crab (lb, 2). The question arises why crystallized calcium carbonates and phosphates do not occur together in calcified tissues. Furthermore, the factors causing the deposition of an amorphous phase in vivo are not known. For some years, we have investigated these problems by studying the effect of phosphate ions on the precipitation and crystallinity of calcium carbonate, and of carbonate ions on those of apatite. In the course of these studies it was found that phosphate ions interfered with the crystallization of calcium carbonate and carbonate ions with that of apatite. Under certain conditions amorphous precipitates of calcium carbonate phosphate were obtained (2). These earlier experiments
AND
125
PHOSPHATES
were made under conditions favoring a rapid spontaneous precipitation at concentrations of calcium, phosphate, and bicarbonate considerably above the physiological range. In the present series of experiments we studied the precipitation of calcium carbonates and phosphates under conditions favoring a slow spontaneous precipitation at concentrations of calcium, phosphate, and bicarbonate and at an ionic strength, pH, and t,emperature within the physiological range. A preliminary report on these studies has been presented elsewhere (13). MATERIALS
AND
METHODS
Solutions. The solutions which were employed in the various series of experiments are listed in Table I. For comparison, the composition of human serum ultrafiltrate with respect to the most important inorganic ions and that of a “standard” calcifying solution used routinely in this laboratory for the in vitro calcification of collagen fibers are also given in the Table. The experimental solutions are grouped in a low bicarbonate (22 mM) and a high bicarbonate (110 mM) series. The low bicarbonate concentra-
TABLE
I
COMPOSITION OF EXPERIMENTAL SOLUTIONS AND OF HUMAN SERUM ULTRAFILTRATE~ mM Human serum tratee Standard tion Low
calcifying
bicarbonate
High bicarbonate
ultrafil-
solu-
series
series
Na
Cl
Cb
136-147
95-131
24-31
145
133
145 145 140-150 230-240 230-240 230-265 260-280
275 225 170 235 230 220 220
230-235 230-235 245-270 250-275
145 130 130 130
a Initial pH of experimental solutions: b Concentration of bicarbonate. c Concentration of phosphate. d Ionic strength. e Values according to Ref. 46b.
7.30 f
22
K 3.4-4.8
5
5 5 22 0
!
I
110
0
I 0.05.
Ca 1.47-1.96
3.75
PC 1.0-1.6
1.67
d 0.16
0.16
75 50 25 12.5 5.0 1.5 1.0
04.667 0-o. 167 O-3.00 O-3.00 o-3.33 O-16.67 O-20.00
0.38 0.30 0.21-0.23 0.26-0.27 0.25-0.26 0.244.27 0.24Xl.30
12.5 5.0 1.5 1.0
O-3.00 O-5.00 O-16.67 O-20.00
0.264.27 0.24-0.25 0.24XJ.25 0.244.32
126
BACHRA,
TRAUTZ,
tion is that of average human serum ultrafiltrate. The high bicarbonate level corresponds to that found in human pancreatic juice. The ionic strength was adjusted with NaCl to the desired value. Potassium chloride was added only to the solutions with t,he highest calcium concentrations. In later experiments it was decided to omit this component from the solutions with the lower calcium concentrations, because potassium ions, present at low concentrations, were not expected to have a significant effect on the results. The solutions were prepared from the following stock solutions of analytical grade reagents: NaCl, 1200; KCl, 50; NaHCOa, 220 or 440; and Na2HPOJ, 167 mM. They were mixed in the required proportions and then diluted with distilled water to about 0.9 of the desired final volume. Carbon dioxide was bubbled through the solutions to lower the pH below 7.0. Then the required amount of a 500 mM CaCL stock solution was added, which had been prepared by dissolving analytical grade CaC08 in dilute HCl, evaporating t.he solution to dryness, dissolving the CaClz in distilled water, and filtering the solution. After bringing the mixed solutions to the desired volume with distilled water the pH was raised to 7.30 f 0.05 by removal of the excess CO* by shaking. Precipitation. After the solutions had been adjusted to a pH of 7.30 + 0.05 they were poured into Erlenmeyer flasks of a-ml. capacity. An air space of at most 2 ml. was left to allow for thermal expansion of the solutions. The flasks were closed with paraffinized cork stoppers held in place by rubber bands and were kept at 37” C. for 5-72 hr. The pH of the supernatant solutions was then determined and their calcium and phosphate concentrations were assayed. The precipitates were quickly washed with distilled water and dried with acet,one. The supernatants and washings were simply poured off when the precipitates adhered to the glass walls. When the precipitates did not adhere they were separated from the fluids by rapid centrifugation. The precipitates were powdered in a mortar for X-ray analysis. Small amounts of the powders were dissolved in dilute HCl for chemical analysis. Chemical analysis. The calcium contents of the supernatant solutions and of the precipitates were determined according to the method of Bachra et al. (14); the phosphate contents were determined according to the method of Fiske and SubbaRow (15). The amounts of precipitate rarely exceeded 10 mg. This made it not feasible to determine their carbonate contents. X-ray analysis. The powder specimens were mounted in Lindemann glass capillaries with a bore of 0.2 mm. and a wall thickness of 0.01 mm. and were placed in a Debye-Scherrer camera of
AND
SIMON
57.4 mm. radius. The exposures with Ni-filtered Cu-radiation at 35 kv. and 20 ma. lasted 6-12 hr. An amorphous precipitate of calcium carbonate phosphate was distinguished from an apatitic precipitate by the absence of the (002) reflection of apatite in the powder patterns. Such patterns also showed appreciable general scatter and, between 27 and 34’ (28)) a diffuse ring instead of the individual apatite lines (112, 211, 300, and 202). RESULTS
In preliminary experiments the standard calcifying solution had been seeded with a very small amount of calcite. After 1 week no measurable amount of precipitate had formed. In similar experiments, in which the solutions had been seeded with apatite, large amounts of an apatitic precipitate had formed in 1 day. This indicated that such a solution is undersaturated with respect to calcite and metastable with respect to apatite. In subsequent experiments we determined what modifications in the composition of this calcifying solution were required for the spontaneous precipitation of crystallized calcium carbonate. The results of these experiments are given in Tables II and III. At the low bicarbonate level of 22 mill (Table II), calcium carbonate was precipitated only when the phosphate concentration of the solution was kept below approximately 0.067 mM and the calcium concentration was higher than approximately 25 mM. When large amounts of calcium carbonate precipitated a considerable drop in the pH occurred (up to 1.3 pH units). At a phosphate concentration of 0.067 mM no precipitate whatsoever formed in 3 days in 8 different experiments at a calcium concentration as high as 75 mM. Thus, at any of the calcium concentrations employed, one could prevent the precipitation of calcium carbonate merely by sufficiently raising the phosphate concentration in the solution. At the high bicarbonate level of 110 mM (Table III) and in the absence of phosphate, a precipitate of calcium carbonate formed already at a calcium concentration of approximately 1.5 mM. Raising the phosphate concentration to 0.167 mM required a raise in the calcium concentration above 5 m?l in order to obtain a precipitate within
TABLE PRECIPITATION
OF CALCIUM
CARBONATES
Initial composition
&f
)
(Af
,
0
75
CT
(c,ad;,
(rs)
63 65 67
0
660
0.067
330
75
0.067
75
0.062 0.054 0.076 <0.050 (0.050 <0.050
220
22
0.167
165
132
75
75
OF 22 MA{
tr
>loo
X-ray
pattrrnc ca + tr ar
ca
tr tr None in 8 expts. 2.94 5.80 2.44 2.47
ca + am
>lOa 15 > 100
0.061 ‘Co.050 <0.050 0.106 0.086 <0.050 <0.050
3.27 12.5 2.71 2.19 2.31 3.94 4.12
0.072 <0.050 0.071 0.063 0.067 0.066
3.25 4.45 2.52 2.58 1.94 7.46 4.62
Or
am + tr ca
am + tr ca Or
am + ca
66
72
(0.050
0.500
44
72
<0.050
6.64
am + ca + tr ar
0.667
33
72
(0.050
4.15
ap (If)
40 41 43
0
cc
0.033
660
0.067
330
0.100
220
10.3
am + ca
0.333
0
22
0
Ca/P (molar)
oc
0.033
0.133
50
c/p
LEVEL
Precipitate
67 6Y 65 60
0.100
AT A BICARBONATE
Final composition of solutions
of s01utmns=
(&
II
ANIJ PHOSPHATES
co
am + ca
+ ca T tr al
ca + tr ar
42 41 42
tr tr tr
50
0.066
50
Cl.067
None in 2 expts.
0.100
None in 1 expt.
50
0.079 0.080 127
> 100
ca
80
ea
2.24 2.57
am + tr ca
TABLE Initial composition of solutions’l
(si,
50
(I&)
22
(A)
0.133
0.167
0
25
22
22
Final composition of solutions
Precipitate
c/p
(I%
Pb (mM)
165
50
0.089 0.083 0.095
7.19 2.15 2.12
50
O.OG6 0.095 0.106 0.077 0.122
2.10 2.53 2.10 2.20 1.99
21 22 23
0
25
0.167
132
m
Ca/P (molar)
m
x-ray patternc
am or am + tr ca
ca + tr ar
0.167
132
0.333
66
24.2 23.4
<0.050 0.090
1.92 2.12
ap (3+)
+ tr ca
0.500
44
23.2 23.1
<0.050 <0.050
2.15 1.73
aP @+I ap (2+)
+ tr ca
0.667
33
21.7 23.2
<0.050 <0.050
2.20 2.06
ap (3+)
+ tr ca
l.OOd
22
21.8 22.1
<0.050 <0.050
2.09 2.47
ap
(3+)
+ tr ca
2.00d
11
20.4
<0.050
2.18
ap (3+)
+ tr ca
18.8
<0.050
1.79
ap (3+)
3.w
12.5
II-Continued
7.3
None in 3 expts.
0 0.167 0.333
13: 66
12.5
0 0.167 0.333
0.500
44
10.7 11.3
0.053 <0.050
2.00 2.11
ap ap
(2+) @+I + tr ca
0.667
33
11.5 10.9
<0.050 <0.050
1.81 2.09
ap
@+I
1.00
22
9.57 10.55
<0.050 0.061
1.83 1.94
2.00
11
8.53
0.079
1.88
7.52
0.090
1.88
5.0
0 0.033 0.167 1.00 1.33
3.00d
7.3
0 0.033 0.167 1.00 1.33
660” 132 22 16.5
128
None in 2 expts.
ap (3-t)
None in 2 expts.
TART,F: Initial romposition of sO1utlOnsa
II---Continued
Final romposition of solutions
1.67
13.2
2.00
11
2.93 3.11
Precipitate
0.345 0.465
1.72 1.75
2.23 2.39
0.310 0.400
1.69 1.6G
1.82 2.08
0.390 0.510
1.68 1.63
1.53 1.59
0.458 0.549
1.46 1.63
ap cl+,
3.00
7.33
1.20 1.38
0.332 0.790
1.61 1.52
3.33
6.60
1.10 1.21
0.391 1.02
1.56 1.71
0
1.5
13.2 6.6 4.4
6.67
3.3
22 8.33
1.0
m
1.67 3.33 5.00
22
0
1.50
1.67 3.33 5.00
None in 2 expts.
1.50
6.67
None in 1 expt.
0.25
5.27
1.50
8.33
1.66
ap (3t) None in 1 expt.
2.6 <0.25
7.03
1.47
8.15 8.25
1.65 1.52
10.00
2.2
13.33
l.ti5
<0.25
11.3
1.67
16.G7
1.32
<0.25
13.8
1.54
0 5.0 6.G7 8.33 10.00 13.33
z4 3.25 2.64 2.2 1.65
1.0
lG.67
1.32
<0.25
15.4
1.66
20.00
1.00
<0.25
17.7
1.58
0 5.0 6.67 8.33 10.00 13.33
ap (3+)
“P (3+)
None in 2 expts.
“P (St) a See Table I for the concentrations of the other ions. Each line in the table represents the results of one individual experiment, unless stated otherwise. The solutions were kept at 37’ C. for 72 hr. The initial pH was 7.30 f 0.05. In general, the final pH was at most a few tenths of a pH unit lower. 110~. ever, when large amounts of calcium carbonate had formed (at 75,50, and 25 mill Ca and low P concentrations) a drop in the pH of up to 1.3 unit occurred. 1 Values obtained by direct analysis of the supernatant solutions. P levels below 0.100 mM and Ca levels below 0.25 m% are subject to errors of 25y0 and more. c ea, calcite; ar, aragonite; am, amorphous; ap, apatite (Its degree of cystallinity is roughly indicated as: (l+), extremely poor; (2-t)) very poor, and (%I-), poor.) tr, trace of. d The solution became turbid when the pH reached 7.0 during the initial pH adjustment, 129
TABLE PRECIFITATION
OF CALCIUM
Initial
CARBONATES
compsition
III
AND PHOSPHATES
AT A BICARBONATE
Final composition of solutions
of solutionsa
T,EVEL
OF 110
Precipitate
X-ray
pattern’
-___ 0
0.167
3.56 0.78 0.75
cc
660
12.5
0.167
10.8
0.100
1.08 8.40
0.333
12.5
5.0
330
0.500
220
O.GG7
167
0
XI
ca + much are ca + some ar’ ca + t,race arg None in 3 expts.”
.> 100
caf
<0.050
>I00
10.9
<0.050
> 100
12.5
0.333
None in 3 expts.
12.5
0.500
None in 3 expts.
12.5
0.667
None in 1 espt.
11.8
0.430
2.34
am + tr ca
12.2 12.3 12.0
0.620 0.687 0.529
2.19 2.26 2.15
am
12.2 10.8
0.720 0.547
2.29 2.17
am + tr ca am
8.80 10.5 10.2 5.25
0.322 0.419 0.387 0.253
3.38 2.18 2.08 4.22
ap (I+) + ca am + tr ca
9.84 4.59 4.57
0.438 0.215 0.226
2.06 5.86 3.57
ap cl+) SP (I+)
ca
-
110
110
1.00
110
1.33
83
l.67d
66
nP (I+)
ap (1-t)
+ ca
aP (lf)
2.OOd
55
2.33d
47
7.46 8.60
0.220 0.290
2.49 2.09
ap @+I + ca + ar al, (2-t)
3.33d
33
4.30
0.200
2.29
“P (3+)
0
cc
0.30
0
0.033
3300
0.51
tr
0.167 0.333 0.667 1.00
660 330 165 110
5.0
0.167 0.333 0.667 1.00
None in 2 expts.
5.0
1.33
None in 2 expts.
1.33
83 4.60
1.16
5.0
1.67
4.7
1.50
1.67
m >lOO
2.00
+ tr ca
ca + tr arf ea
aP (2+)
None in 2 expts.
66
130
+ ca + ca
1.62
ap (a+)
MM
TABLE Initial composition of solutions”
III-Continued
Fina! composition of solutions ._.~
Ca (IIlmM)
C (mW
&I
2.00 5.0
C/P
55
110
X-ray patternC
Ca/P (molar!
(S,
(ZiR,
2.90 2.u 2.52
0.680 0.800 0.607
2.27 2.04
2.15
2.19 1.88 1.78
1.96 “P
2.33
47
2.07 2.11
0.720 0.690 0.797
3.33
33
0.98
1.05
1.93
4.m
27.5
0.485
1.39
1.73
5.00
22
0.403
2.34
1.65
0
02
0.26 0.30 0.35
0
1.67 3.33
60
5.00 6.67
1.5
110
8.33
10.0
33 22 16.5
13.4
11
1.0
8.3
aP (3f)
ca
1.67 1.5
3.33 5.w 6.Gi
None in 3 expts.
None in 1 expt.
1.50
a.33
1.16
7.55 7.60 7.42 7.35
1.66 1.78
8.85 8.45
1.65 1.79
9.52
1.81
1.26 0.29 0.71 <0.25 <0.25 0.82
13.33
cc
@+I
<0.25 0.26
11.7 10.8
1.68
1.70
1.57
1.69
<0.25
14.9
1.65
CO.25 <0.25
14.7 14.5
1.71 1.71)
16.67
6.6
0 5.0 6.67 8.33 10.00
m 22 16.5 13.4
13.33
8.3
0.57
12.3
1.62
16.67
6.6
0.363
15.8
1.62
20.00
5.5
19.0
1.41
1.0
I1
ap (3+)
0 5.0 6.67 8.33
None in 2 expts.
10.00
110
<0.25
aP
(3+)
a See Table I for the concentrations of the other ions. Each line in the Table represents the results of one individual experiment, unless stated otherwise. The solutions were kept at 37’ C., usually for 72 hr., and the initial PH was 7.30 f 0.05. Some experiments were terminated after shorter periods of time (see -0). The final pH had dropped at most a few tenths of a pI1 unit. b-dSee the corresponding legends of Tahle IT. * Experiment terminated after 5 hr. f Experiment terminat.ed after 24 hr. a Experiment terminated after 72 hr. 131
132
HACHRA,
TRAHTZ,
3 days. Less precipitate could form at the high bicarbonate level because lower calcium concentrations were employed. Also, the solutions had a larger buffer capacity. For these reasons, the pH of these solutions dropped at most a few tenths of a pH unit during the precipitation. The precipitation of calcium carbonate was also prevented by trace amounts of pyrophosphate. As little as 4 &’ of sodium pyrophosphate (about 1 ppm) prevented such precipitation at a calcium concentration of 75 mM and a bicarbonate level of 22 mM.
AND
SIMON
The calcium carbonate precipitates formed in the absence of phosphate and kept in contact with the supernatant solution for 3 days consisted of calcite, often mixed with a small amount of aragonite. Examination of the earliest precipitates revealed the presence of considerable amounts of aragonite beside the calcite. Rut during the 3 days contact with the supernatant the aragonite transformed completely or almost completely into calcite. Figure 1 demonstrates such a transformation in the precipitate from a solution containing 12.5 mM calcium, 110 mM bicarbonate, and 0 mM
FIG. 1. Transformation of aragonite into calcite. X-ray patterns of: (a) halibut otolith (pure aragonite) and of precipitates isolated after: (b) 5 hr. (calcite + much aragonite), (c) 24 hr. (calcite + some aragonite), (d) 72 hr. (calcite + trace of aragonite) from a solution containing: 12.5 mn/r Ca, 110 mM bicarbonate and 0 mM phosphate, at p = 0.26,37” C., and pH 7.30 + 0.05.
PRECIPITATION
OF CALCIUM
CARBONATE8
AND PHOSPHATES
133
FIG. 2. X-ray patterns of calcium phosphate precipitates, illustrating the increase in their cystallinity with decreasing C/P ratio of the solution :12.5 mM calcium, 110 mM bicarbonate; see Table III. (a) Amorphous calcium carbonate phosphate (C/P = 83); (b) extremely poorly cystallized apatite + well-crystallized calcite (C/l’ = 66); (c) very poorly crystallized apatite (C/P = 47). phosphate (see Table III). In the presence of phosphate, aragonite did not form at all or only in trace amounts, even in the earliest precipitate. On the other hand, pure aragonite powder, obtained from a halibut otolith, did not transform into calcite when kept in contact with solutions from which no spontaneous precipitation of calcium carbonate or phosphate occurred. These findings indicate that aragonite transformed into calcite only when it was kept in the solutions together with calcite. By raising the phosphate concentration sufficiently, precipitates could again be obtained. These consisted of either amorphous calcium carbonate phosphate or of more or less poorly crystallized apatite., Amorphous precipitates formed at the bicarbonate level of 22 mM, when the C/P ratio was at least 44 and the calcium concentration was > 25 mM. The required minimum C/P ratio and calcium concentra-
tion were 66 and 5 mM, respectively, at the bicarbonate level of 110 mM. The crystallinity of the apatitic precipitates increased with decreasing C/P ratio (see Fig. 2) and decreasing calcium concentration. In many of the precipitates larger or smaller amounts of calcite and, occasionally, traces of aragonite were found beside the amorphous phase or the apatite. Such crystallized calcium carbonates appeared when the phosphate concentration had diminished considerably during the precipitation. 11ISCUSSION
THE EFFECT OF PHOSPHATE ON THE PRECIPITATION OF CALCIUM CARBOKATE The results of our experiments show that, under physiological conditions, the spontaneous precipitation of calcium carbonate
13-l
BACHRA,
TRAUTZ,
can be prevented by the presence of low concentrations of phosphate ions. At both bicarbonat,e levels such precipitation was prevented when the phosphate/bicarbonate concentration ratio was higher than about l/300. Above this ratio, the competition of the phosphate ions with the carbonate ions for the calcium ions was evidently sufficient to inhibit the nucleation and crystal growth of calcite and aragonite, while the phosphate concentration was still too low for the nucleation of calcium phosphate. Pyrophosphate and hexametaphosphate inhibit the precipitation of calcium carbonate in a similar way (16-19). Table IV shows the range of concentrations of some of the most import#ant ions present in a number of biological fluids, as reported in the literature. Our findings indicate that calcium carbonate deposition should not take place in these fluids on account of the inhibitory effect of the phosphate ions. Even when solutions similar in composition to these biological fluids were seeded with calcite, no precipitation of additional calcite was obtained (20). The composition of the endolymph in the
COMPOSITION
OF SOME BIOLOGICAL
mM
Na
Cl
ANI)
human inner ear, as reported in the literature (see Table IV), is similar to that of serum ultrafiltrate. This suggests that the human statoconia, which consist of calcite, are deposited in a fluid which differs in composition from endolymph. Our chemical analysis of a halibut otolith, which gave the X-ray patt,ern of pure aragonite, showed no detectable phosphate. The phosphate ions would have been coprecipitated with the calcium carbonate, had any been present in the fluid at the site of otolith formation (see Tables II and III), since the phosphate ions are bound more strongly to the calcium ions than are the carbonate ions. We postulate therefore that such otoliths are formed in a solution essentially free of phosphate ions. A report in the literature (21) states that calcite calculi have been found in the pancreatic tissue of a patient suffering from chronic pancreatitis. In this connection, it is of interest that pancreatic juice contains bicarbonate in large concentrations and can be very low in phosphate. Calcium carbonate has not been found in urinary calculi (22, 23). This is most likely
TABLE IV FLUIDS WITH RESPECT K
c8.a
SIMON
TO THE MOST
IMPORTANT
Pb
CC
Msa
INORGANIC
IONS
PH
Reid
_-
mammalian serum
136-171
90-156
3.4-10.2
1.8-5.0
0.5-1.8
0.7-3.0
-
4.6-6.4
3.3-5.9 -__
0.45-2.55
-
102-160
2.4-7.9 ~---
2.04.1
0.58-0.99 --__ 0.5-7.9
0.7-5.8
0.08-0.53 --__
2.44.8 __------__-_-_
2-48 ~--~__-__ 0.57-3.2
0.15
0.008&0.41
1630
7.3-7.4
46b
-----------__ bird
serum ----__-__--
reptilian --
serum
loo-186 -----__
human saliva _~____
3.5-57.98.4-17.712.7-37.80.9-2.4 ~---__ --------
human pancrea113-153 tic juice ---~-~---____~human lymph ---__ crustacean olymph
endo-
6-30
54-95
2.6-7.4
1.1-1.6
loo-123
128-169
0.7-1.45
56-590
180-1690
0.546
46c -___
-
46d
5.6-7.6
46e
66-127
8.6-8.8
46f
7.7-14.5
-
~_-------__--__-_
-----------
hem-
-__-
1.2-1.3
0.30.7
47, 48
-~------------__-__-______-_ 5.342.5
2.5-53
0.164.48
5.5-32.57.5-8.5
0 Only part of the calcium and magnesium is present in ionic form. b Phosphorus present as inorganic phosphate. c Bicarbonate. d The concentration ranges of the ions were derived from the values tabulated
in the cited
29b,46a, and 49
references.
PRECIPITATIOX
OF CALCIUM
CARBONATES
AND
PHOSPHATES
135
ways to deposit fine-grained calcite in COIlagenous or chitinous fibrous matrix but were not successful in obtaining such deposits (31). This indicates that calcite deposition in the tissues of these arthropods occurs under conditions which we have not yet been able to duplicate in the laboratory. The simultaneous growth of a large number of nuclei in a small volume of tissue may give rise to a multitude of very small crystallites. The smallness of such crystallites may be due in part also to impurities, like phosphate and pyrophosphate, which at higher concentrations, inhibit the precipitation of calcium carbonate, and at lower coneentrations disturb its crystallization to such an extent that the growth of the crystallites into larger crystals is prevented (16-19). In our experiments, however, we never observed such fine-grained deposits, even when phosphate or pyrophosphate was present. Such deposits may have escaped our attention, because the bulk of the phosphate and pyrophosphate is removed from the solution with the precipitate at an early stage of the precipitation. The large quantities of wellcrystallized material formed after the removal of the phosphate or pyrophosphate may have masked the fine-grained fraction on the X-ray patterns. Biological deposits of pure aragonite are found in the otoliths of bone fishes (6a, 7-10) and in the skeletons of many invertebrates (6b). The results of our experiments indicate that deposits of calcite-free aragonite do not easily transform into calcite under physiological conditions. Yo adequate explanation, however, is available at present for the frequent biological deposition of metastable aragonite in preference to the stable calcite, though it is known that such deposition is SPECIAL FEATURES 0~ SOME BIOLOGIC~~L facilitated by elevated temperature (32-34), CALCIUM CARBONATES by certain metal ions (35), and by epitactic fact’ors (36). That epitactic factors can cause The calcite and aragonite precipitates the deposition of metastable instead of the obtained in our experiments were always well crystallized and coarse-grained, as stable modification has been shown also shown by t.he spotty appearance of the with other salts (37, 38). A third modification of calcium carbonate, X-ray reflections. We never obtained crysvaterite, has been demonstrated in the tallites as small as the very fine-grained calcite deposited in vivo in the carapace and calcified tissues of certain invertebrates tendon plates of certain arthropods (1~). (6b). The factors causing its deposition in the tissues are at present unknown. In other studies we att,empted in various
due to the high phosphate concentration in this fluid. The composition of human saliva precludes the crystallization of calcium carbonate in salivary and dental calculi (2426). The presence of calcite in dental calculus from the horse, dog, ferret, and hamster (27) makes it of interest to study the composition of the saliva in these animals. In some crustaceans the hemolymph has a composition similar to that of our experimental solutions from which amorphous calcium carbonate phosphate precipitated. Small amounts of such amorphous deposits have, indeed, been found in the skeletons of some crustaceans (lb, lc, 2). The principal mineral deposit in these tissues, however, consists of calcite. One would not expect such deposits to form in the hemolymph, since its phosphate concentration is too high to allow this. On the basis of the above considerations we feel that cellular activity may be required to modify the composition of the tissue fluid at the sites of calcium carbonate deposition in most tissues where such deposits occur. For instance, the cells could provide a raised carbonate level. Large amounts of COZ may be formed by metabolic processes and be rapidly hydrated under the influence of carbonic anhydrase. This enzyme was recently demonstrated in exceptionally large quantities in the inner ear of the cat (28). It also occurs in high concentrations in the oviducts of birds (llb) and in the calcium carbonate depositing tissues of many invertebrates (29a, 30). The phosphate concentration may be lowered at the sites of calcium carbonate deposition by metabolic processes favoring the formation of phosphate esters.
136
BACHRA.
TRAUTZ,
THE EFFECT OF CARBONATE ON THE PRECIPITATION OF CALCIUM PHOSPHATE
AND
SIMON
of such amorphous precipitates is also governed by some sort of an ion product relationship. The final Ca and P concentrations in the In earlier experiments (2) it was found solution after 3 days (Tables II and III) that the presence of carbonate in the soluwere higher when the precipitate was amortion reduced the crystallinity of the precipitated apatite. These experiments were made phous than when it was apat,itic. They also were higher in the experiments at the high under conditions favoring rapid spontaneous than at the low bicarbonate level. This indiprecipitation at concentrations of calcium, cates t,hat the presence of bicarbonate inphosphate, and bicarbonate considerably above the physiological range. An amor- creases the solubility of the solid phase and is more phous calcium carbonate phosphate was that an amorphous precipitate soluble than an apatitic one. It must be obtained when the C/P molar ratio in the borne in mind, however, that the precipisolution was 17 or higher. The precipitate tated solids and the supernatant solutions gradually changed into a poorly crystallized were usually not yet in equilibrium after apatite when it was kept in contact with its 3 days. During the deposition of larger supernatant solution for several days. In the present series of experiments condi- amounts of precipitate the composition of the solution changes markedly and with it tions were employed favoring a slow precipitation at physiological concent,rations and also the composition and character of the precipitating solid. For instance, calcite pH. The amorphous precipitates obtained under these conditions were found to be frequently crystallized after the amorphous stable when kept in contact with the super- or apatitic precipitate formed earlier had removed enough phosphate from the solunatant solution for 3 days, but the minimum tion to raise the C/P ratio sufficiently. Also, C/P ratio required was considerably higher: when an amorphous precipitate is depositing, 44 at 22 mM bicarbonate, and 66 at 110 mM bicarbonate. Evidently, the carbonate ions the calcium concentration in the solution compete with the phosphate ions for the will decrease and this may enable the available calcium on the surface of the solid deposition of an apatitic precipitate at a later stage (20). phase and, at a sufficiently high C/P ratio, The chemical analysis of the amorphous can prevent the orderly crystallization of the apatite. Such competition is more effec- calcium carbonate phosphat,es, which contive when the precipitation is rapid and a tained no or only a trace of calcite according lower C/P ratio suffices for the formation of to the X-ray pattern, showed Ca/P molar an amorphous precipitate. ratios of about 2.1-2.3, while the theoretical As the phosphate is bound more strongly value for apatite is 1.67. The apatitic preto the calcium than is the carbonate, a great cipitates had Ca/P ratios ranging from 2.2 excess of carbonate in the solution is re- to 1.4. Apatitic precipitates with a high quired to prevent the crystallization of apa- Ca/P ratio were usually obtained from solutite and to cause the formation of an amor- tions with a high C/P ratio. They were phous precipitate. For the same reason, the poorly crystallized and probably conbained C/P ratio in the amorphous precipitate is also a large amount of an amorphous phase alwa,ys many times (1 to 2 orders of magnitude) lower than the C/P ratio in the solu- (20). The higher Ca/P ratios of the amorphous precipitates indicate that, at least, tion from which it was formed (Id). At a particular C/P ratio, the lower P 20-27s of the Ca ions in such material are (and C) concentrations required higher Ca bound to carbonate. The actual percentage should even be higher than that, since conconcentrations for the spontaneous precipisiderable amounts of HP04 and Na ions are tation of an amorphous calcium carbonate phosphate than the higher P (and C) con- also present, which tend to lower the Ca/P ratio of the precipitates. centrations. This suggests that the formation
PRECIPITATION
OF CALCIUM
CARBONATES
THE EFFECT OF T~VPURITIES ON THE CRYSTALLINITY OF THE BIOLOGICAL APAT~TES
The amorphous precipitates of calcium carbonate phosphate found in the carapace and tendon plates of certain arthropods and in the concretions of tapeworms (lb, lc, 2, 12) may form under condit,ions similar to those employed in our experiments. The Ca/P ratios in the tapeworm concretions are higher than 3 (12). We feel that these biological depo,sits, in spite of their high Ca/P ratios, are completely amorphous on account of the presence of magnesium. Experiments to be reported (20) show that the presence of magnesium ions facilitates the formation of amorphous calcium carbonate phosphate and slows down or prevents its crystallization into apatite. The apatite crystallites of calcified tissues are very small. In bone (39, 40), dentin (41, 42), and calcified cartilage (43), platelets and needles have been found with dimensions varying between less th$n 100 and somew.hat more than 1000 A. Our results suggest that the smallness of these crystallites is due to the presence of impurities, like HPO,, carbonate and magnesium ions, which cause lattice distortion and limit the crystal size. The apatite crystals in dental enamel (le, 44, 45) are larger and better crystallized than those in t,he ot.her mineralized tissues. They contain less of the various impurities (Mg, Ka, COa, HPO+ citrate, etc.). It is likely therefore that the ameloblasts modify the tissue fluid in which these crystals form, so that it contains less of these impurities than the tissue fluid in bone and dentin. ACKNOWLEDGMENT We acknowledge the assistance of Mr. Conetta in the early phases of this work.
A. R.
REFERENCES 1. TRAUTZ, 0. R., (a) Ann. A:. Y. Acad. Sci. 60, 696 (1955); (b) ibid. 86, 145 (1960); (c) Progress Reports Contract DA-49.007.MD-765, Research & Development Division, Office of the Surgeon General, U. S. Army, 1957, 1958 and 1959; (d) unpublished results, (e) Ann. Dent. 12, 47 (1953).
AND
PHOSPHATES
137
2. TRAUTZ, 0. R., AND ZAPANTA, R. R., Arch. Oral Bid. 4, 122, (1961). 3. CARLSTROM, D., AND ENGSTR~M, A., In “The Biochemistry and Physiology of Bone,” (G. H. Bourne, ed.), Chapter 6. Academic Press, New York, 1956. 4. NEUMAN, W. F., AND NEUMAN, M. W., Chew ical Revs. 63, 1 (1953) and “The Chemical Dynamics of Bone Mineral,” Univ. Chicago Press, 1958. Suppl. 121 5. CARLSTRBM, D., Acta Radiol., (1955). 6. VINOGRADOV, A. P., “The Elementary-Chemical Composition of Marine Organisms” (Translated by J. Efron and J. K. Setlow with bibliography by V. W. Odum), Memoir No. II of the Sears Foundation for Marine Research, Yale University, New Haven, 1953; (a) p. 561, (b) Chapter 20. 7. CARLSTRGM, D., ENGSTR~M, A., AND HJORTH, S., Laryngoscope 63, 1052 (1953). 8. CARLSTRGM, D., AND ENGSTR~M, A., Acta Oto-Laryng. 46, 12 (1955). 9. SASAKI, H., AND MIYATA, J., Z. Laryngol. Rhinol. Otol. 34, 740 (1955). 10. IRIE, T., J. Fat. Fisheries Animal Husbandry Hiroshima Univ. 1,l (1955) and 3,203 (1960). 11. ROMANOFF, A. L., AND ROMANOFF, A. J., “The Avian Egg,” (a) p. 353, (b) p. 226. Wiley, New York, 1949. 12. VON BRAND, T., MERCADO, T. I., NYLEN, M. U., AND SCOTT, 1). B., Exptl. Parasitol. 9, 205 (1960). 13. BACHRA, B. N., TRAUTZ, 0. R., AND CONETTA, A. R., “Precipitation of Calcium CarbonTransactions Internat. ate Phosphate,” Assoc. for Dent. Res., 40th General Meeting 1962, Abstract A 102. 14. BACHRA, B. S., DAVER, A., AND SOBEL, A. E., Clin. Chem. 4, 107 (1958). 15. FISKE, C. H., AND SUBBAROW, Y., J. Riol. Chem. 66, 275 (1925). 16. HATCH, G. B., AND RICE, O., Znd. Eng. Chem 31, 51 (1939). R. F., AND BUEHRER, T. F., 17. REITEMEIER, J. Phys. Chem. 44, 535 (1940). 18. Br-EHRER, T. F., AND REITEMEIER, R. F., J. Phys. Chem. 44, 552 (1940). Faraday SOC. 6, 19. RAISTRICK, B., Discussions 234 (1949). 20. BACHRA, B. N., TRAUTZ, 0. R., AND SIMON, S. L., In preparation. 21. PARSONS, J., AND EURS, F. J., Am. J. Clin. Pathol. 6, 405 (1959). 22. PRIEN, E. L., AND FRONDEL, C., J. Ural. 6’7, 949 (1947).
138
BACHRA,
TRAUTZ,
23. LAGERGREN, C., Acta Radiol. Suppl. 133 (1956). 24. JENSEN, A. T., AND DANO, M., J. Dental Res. 31, 620 (1952) and 33, 741 (1954). 25. JENSEN, A. T., AND HANSEN, K. G., Experientia 16, 311 (1957). 26. JENSEN, A. T., AND ROWLES, S. L., Acta Odont. Stand. 16, 121 (1957) and Xature 179, 912 (1957). 27. UNMACK, A., AND ROWLES, S. L., Nature 197, 486 (1963). 28. ERULKAR, S. D., AND MAREN, T. H., Nature 189, 459 (1960). 29. TRAVIS, D. F., (a) p. 57 in “Calcification in Biological Systems” (R. F. Sognnaes, ed.), Publication No. 64 of the A. A. A. S., Washington, D. C., 1960; (b) Biol. Bull. 109, 484 (1955) and 113, 451 (1957). 30. WILBUR, K. M., p. 15 in “Calcification in Biological Systems” (R. F. Sognnaes, ed.), Publication No. 64 of the A. A. A. S., Washington, D. C., 1960. 31. TRAUTZ, 0. R., AND BACHRA, B. N., Arch. Oral Biol., in press. 32. LOWENSTAM, H. A., Proc. Natl. Acad. Sci. 40, 39 (1954); J. Geol. 62, 284 (1954); and J. Sed. Petrol. 26, 270 (1955). 33. ZELLER, E. J., ANU WRAY, J. L., Bull. Am. Assoc. Petrol. Geol. 40, 140 (1956). 34. WRAY, J. L., AND DANIELS, F., J. Am. Chem. Sot. 79, 2031 (1957). 35. MURRAY, J. W., J. Geol. 62, 481 (1954). 36. WATABE, N., AND WILBUR, K. M., Nature 188, 334 (1960).
AND
SIMON
37. STRANSKI, I. N., AND ML-TAFTSCHIEW, Z. C., Ztschr. Physik. Chemie A160, 135 (1930). 38. SCHULZ, L. G., Acta Cry&. 6, 266 (1952). 39. ROBINSON, R. A., AND WATSON, M. L., Ann. N. Y. Acad. Sci. 60, 596 (1955). 40. FERNANDEZ-MORAN, H., AXD ENGSTRijM, A., Biochim. Biophys. Acta 23, 260 (1957). 41. JENSEN, A. T., AND MOELLER, A., J. Dental Res. 27, 524 (1948). 42. CARLSTR~~M, D., AND G~as, J. E., Biochim. Biophys. Acta 36, 46 (1959). 43. DURNING, W. C., J. Ultrastruct. Res. 2, 245 (1958). 44. TRAIJTZ, 0. R., KLEIN, E., FESSENDEN, E., AND ADDELSTON, H. K., J. Denfal Res. 32, 420 (1953). 45. GLAS, J. E., AND OMNELL, K. A., J. C%rastruct. Res. 3, 334 (1960). 46. ALTMAN, P. L., “Blood and Other Body Fluids,” Biological Handbooks Series, Fed. Am. Sot. Exptl. Biol., Washington, D. C., 1961, (a) Table 100, Part IV (pp. 294-299); (b) Tables 6 (p. 22), 7 part III (p. 27), and 8 part III (p. 35); (c) Table 9 (p. 37); (d) Table 10 (p. 39); (e) Table 130 part I (p. 399); (f) Table 134 part I (p. 414); and (g) Table 122 part I (p. 363). 47. CITRON, L., AND EXLEY, D., Proc. Roy. Sot. Med. 60, 697 (1957). 48. RATCH, S., Z. Laryngol. Rhino/. Otol. 39, 16 (1960); and Arkiv Ohren usw. Heilk. Z. Hals- usw. Heilk. 178, 126 (1961). 49. HAYES, 1). K., SINGER, L., ANU ARMSTRONG, W. r)., Proc. Sot. Exptl. Biol. Med. 109, 126 (1962).
OF
BIOCHEMISTRY
Precipitation
AND
BIOPHYSICS
of Calcium
Precipitation
124-138 (1963)
103,
Carbonates
of Calcium
and
Carbonates
Physiological BERNARD From the Department
N. BACHRA, of Biochemistry
OTTO
Phosphates. and
Phosphates
under
Conditions’
R. TRAUTZ
AND
S. LAWRENCE
and Guggenheim Institute for Dental College of Dentistry, New York Received
I. Spontaneous
Research,
SIMON
h’ew York University
June 12, 1963
The slow spontaneous precipitation of calcium carbonates and calcium phosphates was studied under physiological conditions. Calcium carbonate precipitation was prevented by the presence of phosphate ions at concentrations too low for the precipitation of calcium phosphate. The composition of most biological fluids does not allow the deposition of calcium carbonate. The suggestion is made that such deposition will occur under the influence of metabolic processes which locally raise the bicarbonate and lower the phosphate concentration. Aragonite was metastable under the conditions of the experiments. The aragonite present in the early precipitates of calcium carbonate transformed into calcite, when kept in contact with the supernatant solution. The crystallization of apatite is disturbed when bicarbonate is present in the solution. At sufficiently elevated calcium concentrations and bicarbonate/phosphate concentration ratios, an amorphous precipitate of calcium carbonate phosphate formed, which failed to crystallize into apatite when kept in contact with the supernatant solution. The Ca/P molar ratio of the apatitic and amorphous precipitates varied between approximately 2.3 and 1.4. This large variation is due to the coprecipitation of carbonate, HPOa, and Na ions. These and other impurities interfere with the crystallization of the biological apatites and limit the size of the crystallites in bone and dentine. Dental enamel is better crystallized because it contains lesser amounts of these impurities.
tites has been ascribed to the presence of these impurities (la, lb, 2). Crystallized calcium carbonate does not occur in the bone and teeth of the vertebrates (la, 3-5). It is found in this phylum in specific tissues only and no apatite occurs with it. Thus the otoliths in fishes and the statoconia in amphibia contain aragonite (6a, 7-lo), while the statoconia in mammals (6a, 7-9) and the egg shells of birds (lla) and reptiles (Id) contain calcite. The calcified tissues of the invertebrates usually contain calcium carbonate (calcite, aragonite, or vaterite) as the mineral phase. Apatite is rarely found in the invertebrates (6b).
INTRODUCTION
The mineral phase of bone and teeth in the vertebrates gives the X-ray pattern of an apatite. It is a basic calcium phosphate, containing varying amounts of impurities, like carbonate, secondary phosphate, citrate, magnesium, sodium, and potassium. The poor crystallinity of many biological apaI This work was supported in part by a research grant, D-159, from the National Institute of Dental Research, U. S. Public Health Service, and by the Research and Development Command, Office of the Surgeon General Department of the U. S. Army, under Contract No. DA-49.907-MD765. 24
PRECIPITATION
OF CALCIUM
CARBONATES
In some organisms the crystallisat.ion of the mineral phase is disturbed to such a degree that it is completely amorphous. Such amorphous material was demonstrated in small concretions in the tapeworm Taenia taeniaejormis and Cysticewus jasciolaris (lb, lc, 2, 12) and in the carapace and tendon plates of the mantis shrimp and stone crab (lb, 2). The question arises why crystallized calcium carbonates and phosphates do not occur together in calcified tissues. Furthermore, the factors causing the deposition of an amorphous phase in vivo are not known. For some years, we have investigated these problems by studying the effect of phosphate ions on the precipitation and crystallinity of calcium carbonate, and of carbonate ions on those of apatite. In the course of these studies it was found that phosphate ions interfered with the crystallization of calcium carbonate and carbonate ions with that of apatite. Under certain conditions amorphous precipitates of calcium carbonate phosphate were obtained (2). These earlier experiments
AND
125
PHOSPHATES
were made under conditions favoring a rapid spontaneous precipitation at concentrations of calcium, phosphate, and bicarbonate considerably above the physiological range. In the present series of experiments we studied the precipitation of calcium carbonates and phosphates under conditions favoring a slow spontaneous precipitation at concentrations of calcium, phosphate, and bicarbonate and at an ionic strength, pH, and t,emperature within the physiological range. A preliminary report on these studies has been presented elsewhere (13). MATERIALS
AND
METHODS
Solutions. The solutions which were employed in the various series of experiments are listed in Table I. For comparison, the composition of human serum ultrafiltrate with respect to the most important inorganic ions and that of a “standard” calcifying solution used routinely in this laboratory for the in vitro calcification of collagen fibers are also given in the Table. The experimental solutions are grouped in a low bicarbonate (22 mM) and a high bicarbonate (110 mM) series. The low bicarbonate concentra-
TABLE
I
COMPOSITION OF EXPERIMENTAL SOLUTIONS AND OF HUMAN SERUM ULTRAFILTRATE~ mM Human serum tratee Standard tion Low
calcifying
bicarbonate
High bicarbonate
ultrafil-
solu-
series
series
Na
Cl
Cb
136-147
95-131
24-31
145
133
145 145 140-150 230-240 230-240 230-265 260-280
275 225 170 235 230 220 220
230-235 230-235 245-270 250-275
145 130 130 130
a Initial pH of experimental solutions: b Concentration of bicarbonate. c Concentration of phosphate. d Ionic strength. e Values according to Ref. 46b.
7.30 f
22
K 3.4-4.8
5
5 5 22 0
!
I
110
0
I 0.05.
Ca 1.47-1.96
3.75
PC 1.0-1.6
1.67
d 0.16
0.16
75 50 25 12.5 5.0 1.5 1.0
04.667 0-o. 167 O-3.00 O-3.00 o-3.33 O-16.67 O-20.00
0.38 0.30 0.21-0.23 0.26-0.27 0.25-0.26 0.244.27 0.24Xl.30
12.5 5.0 1.5 1.0
O-3.00 O-5.00 O-16.67 O-20.00
0.264.27 0.24-0.25 0.24XJ.25 0.244.32
126
BACHRA,
TRAUTZ,
tion is that of average human serum ultrafiltrate. The high bicarbonate level corresponds to that found in human pancreatic juice. The ionic strength was adjusted with NaCl to the desired value. Potassium chloride was added only to the solutions with t,he highest calcium concentrations. In later experiments it was decided to omit this component from the solutions with the lower calcium concentrations, because potassium ions, present at low concentrations, were not expected to have a significant effect on the results. The solutions were prepared from the following stock solutions of analytical grade reagents: NaCl, 1200; KCl, 50; NaHCOa, 220 or 440; and Na2HPOJ, 167 mM. They were mixed in the required proportions and then diluted with distilled water to about 0.9 of the desired final volume. Carbon dioxide was bubbled through the solutions to lower the pH below 7.0. Then the required amount of a 500 mM CaCL stock solution was added, which had been prepared by dissolving analytical grade CaC08 in dilute HCl, evaporating t.he solution to dryness, dissolving the CaClz in distilled water, and filtering the solution. After bringing the mixed solutions to the desired volume with distilled water the pH was raised to 7.30 f 0.05 by removal of the excess CO* by shaking. Precipitation. After the solutions had been adjusted to a pH of 7.30 + 0.05 they were poured into Erlenmeyer flasks of a-ml. capacity. An air space of at most 2 ml. was left to allow for thermal expansion of the solutions. The flasks were closed with paraffinized cork stoppers held in place by rubber bands and were kept at 37” C. for 5-72 hr. The pH of the supernatant solutions was then determined and their calcium and phosphate concentrations were assayed. The precipitates were quickly washed with distilled water and dried with acet,one. The supernatants and washings were simply poured off when the precipitates adhered to the glass walls. When the precipitates did not adhere they were separated from the fluids by rapid centrifugation. The precipitates were powdered in a mortar for X-ray analysis. Small amounts of the powders were dissolved in dilute HCl for chemical analysis. Chemical analysis. The calcium contents of the supernatant solutions and of the precipitates were determined according to the method of Bachra et al. (14); the phosphate contents were determined according to the method of Fiske and SubbaRow (15). The amounts of precipitate rarely exceeded 10 mg. This made it not feasible to determine their carbonate contents. X-ray analysis. The powder specimens were mounted in Lindemann glass capillaries with a bore of 0.2 mm. and a wall thickness of 0.01 mm. and were placed in a Debye-Scherrer camera of
AND
SIMON
57.4 mm. radius. The exposures with Ni-filtered Cu-radiation at 35 kv. and 20 ma. lasted 6-12 hr. An amorphous precipitate of calcium carbonate phosphate was distinguished from an apatitic precipitate by the absence of the (002) reflection of apatite in the powder patterns. Such patterns also showed appreciable general scatter and, between 27 and 34’ (28)) a diffuse ring instead of the individual apatite lines (112, 211, 300, and 202). RESULTS
In preliminary experiments the standard calcifying solution had been seeded with a very small amount of calcite. After 1 week no measurable amount of precipitate had formed. In similar experiments, in which the solutions had been seeded with apatite, large amounts of an apatitic precipitate had formed in 1 day. This indicated that such a solution is undersaturated with respect to calcite and metastable with respect to apatite. In subsequent experiments we determined what modifications in the composition of this calcifying solution were required for the spontaneous precipitation of crystallized calcium carbonate. The results of these experiments are given in Tables II and III. At the low bicarbonate level of 22 mill (Table II), calcium carbonate was precipitated only when the phosphate concentration of the solution was kept below approximately 0.067 mM and the calcium concentration was higher than approximately 25 mM. When large amounts of calcium carbonate precipitated a considerable drop in the pH occurred (up to 1.3 pH units). At a phosphate concentration of 0.067 mM no precipitate whatsoever formed in 3 days in 8 different experiments at a calcium concentration as high as 75 mM. Thus, at any of the calcium concentrations employed, one could prevent the precipitation of calcium carbonate merely by sufficiently raising the phosphate concentration in the solution. At the high bicarbonate level of 110 mM (Table III) and in the absence of phosphate, a precipitate of calcium carbonate formed already at a calcium concentration of approximately 1.5 mM. Raising the phosphate concentration to 0.167 mM required a raise in the calcium concentration above 5 m?l in order to obtain a precipitate within
TABLE PRECIPITATION
OF CALCIUM
CARBONATES
Initial composition
&f
)
(Af
,
0
75
CT
(c,ad;,
(rs)
63 65 67
0
660
0.067
330
75
0.067
75
0.062 0.054 0.076 <0.050 (0.050 <0.050
220
22
0.167
165
132
75
75
OF 22 MA{
tr
>loo
X-ray
pattrrnc ca + tr ar
ca
tr tr None in 8 expts. 2.94 5.80 2.44 2.47
ca + am
>lOa 15 > 100
0.061 ‘Co.050 <0.050 0.106 0.086 <0.050 <0.050
3.27 12.5 2.71 2.19 2.31 3.94 4.12
0.072 <0.050 0.071 0.063 0.067 0.066
3.25 4.45 2.52 2.58 1.94 7.46 4.62
Or
am + tr ca
am + tr ca Or
am + ca
66
72
(0.050
0.500
44
72
<0.050
6.64
am + ca + tr ar
0.667
33
72
(0.050
4.15
ap (If)
40 41 43
0
cc
0.033
660
0.067
330
0.100
220
10.3
am + ca
0.333
0
22
0
Ca/P (molar)
oc
0.033
0.133
50
c/p
LEVEL
Precipitate
67 6Y 65 60
0.100
AT A BICARBONATE
Final composition of solutions
of s01utmns=
(&
II
ANIJ PHOSPHATES
co
am + ca
+ ca T tr al
ca + tr ar
42 41 42
tr tr tr
50
0.066
50
Cl.067
None in 2 expts.
0.100
None in 1 expt.
50
0.079 0.080 127
> 100
ca
80
ea
2.24 2.57
am + tr ca
TABLE Initial composition of solutions’l
(si,
50
(I&)
22
(A)
0.133
0.167
0
25
22
22
Final composition of solutions
Precipitate
c/p
(I%
Pb (mM)
165
50
0.089 0.083 0.095
7.19 2.15 2.12
50
O.OG6 0.095 0.106 0.077 0.122
2.10 2.53 2.10 2.20 1.99
21 22 23
0
25
0.167
132
m
Ca/P (molar)
m
x-ray patternc
am or am + tr ca
ca + tr ar
0.167
132
0.333
66
24.2 23.4
<0.050 0.090
1.92 2.12
ap (3+)
+ tr ca
0.500
44
23.2 23.1
<0.050 <0.050
2.15 1.73
aP @+I ap (2+)
+ tr ca
0.667
33
21.7 23.2
<0.050 <0.050
2.20 2.06
ap (3+)
+ tr ca
l.OOd
22
21.8 22.1
<0.050 <0.050
2.09 2.47
ap
(3+)
+ tr ca
2.00d
11
20.4
<0.050
2.18
ap (3+)
+ tr ca
18.8
<0.050
1.79
ap (3+)
3.w
12.5
II-Continued
7.3
None in 3 expts.
0 0.167 0.333
13: 66
12.5
0 0.167 0.333
0.500
44
10.7 11.3
0.053 <0.050
2.00 2.11
ap ap
(2+) @+I + tr ca
0.667
33
11.5 10.9
<0.050 <0.050
1.81 2.09
ap
@+I
1.00
22
9.57 10.55
<0.050 0.061
1.83 1.94
2.00
11
8.53
0.079
1.88
7.52
0.090
1.88
5.0
0 0.033 0.167 1.00 1.33
3.00d
7.3
0 0.033 0.167 1.00 1.33
660” 132 22 16.5
128
None in 2 expts.
ap (3-t)
None in 2 expts.
TART,F: Initial romposition of sO1utlOnsa
II---Continued
Final romposition of solutions
1.67
13.2
2.00
11
2.93 3.11
Precipitate
0.345 0.465
1.72 1.75
2.23 2.39
0.310 0.400
1.69 1.6G
1.82 2.08
0.390 0.510
1.68 1.63
1.53 1.59
0.458 0.549
1.46 1.63
ap cl+,
3.00
7.33
1.20 1.38
0.332 0.790
1.61 1.52
3.33
6.60
1.10 1.21
0.391 1.02
1.56 1.71
0
1.5
13.2 6.6 4.4
6.67
3.3
22 8.33
1.0
m
1.67 3.33 5.00
22
0
1.50
1.67 3.33 5.00
None in 2 expts.
1.50
6.67
None in 1 expt.
0.25
5.27
1.50
8.33
1.66
ap (3t) None in 1 expt.
2.6 <0.25
7.03
1.47
8.15 8.25
1.65 1.52
10.00
2.2
13.33
l.ti5
<0.25
11.3
1.67
16.G7
1.32
<0.25
13.8
1.54
0 5.0 6.G7 8.33 10.00 13.33
z4 3.25 2.64 2.2 1.65
1.0
lG.67
1.32
<0.25
15.4
1.66
20.00
1.00
<0.25
17.7
1.58
0 5.0 6.67 8.33 10.00 13.33
ap (3+)
“P (3+)
None in 2 expts.
“P (St) a See Table I for the concentrations of the other ions. Each line in the table represents the results of one individual experiment, unless stated otherwise. The solutions were kept at 37’ C. for 72 hr. The initial pH was 7.30 f 0.05. In general, the final pH was at most a few tenths of a pH unit lower. 110~. ever, when large amounts of calcium carbonate had formed (at 75,50, and 25 mill Ca and low P concentrations) a drop in the pH of up to 1.3 unit occurred. 1 Values obtained by direct analysis of the supernatant solutions. P levels below 0.100 mM and Ca levels below 0.25 m% are subject to errors of 25y0 and more. c ea, calcite; ar, aragonite; am, amorphous; ap, apatite (Its degree of cystallinity is roughly indicated as: (l+), extremely poor; (2-t)) very poor, and (%I-), poor.) tr, trace of. d The solution became turbid when the pH reached 7.0 during the initial pH adjustment, 129
TABLE PRECIFITATION
OF CALCIUM
Initial
CARBONATES
compsition
III
AND PHOSPHATES
AT A BICARBONATE
Final composition of solutions
of solutionsa
T,EVEL
OF 110
Precipitate
X-ray
pattern’
-___ 0
0.167
3.56 0.78 0.75
cc
660
12.5
0.167
10.8
0.100
1.08 8.40
0.333
12.5
5.0
330
0.500
220
O.GG7
167
0
XI
ca + much are ca + some ar’ ca + t,race arg None in 3 expts.”
.> 100
caf
<0.050
>I00
10.9
<0.050
> 100
12.5
0.333
None in 3 expts.
12.5
0.500
None in 3 expts.
12.5
0.667
None in 1 espt.
11.8
0.430
2.34
am + tr ca
12.2 12.3 12.0
0.620 0.687 0.529
2.19 2.26 2.15
am
12.2 10.8
0.720 0.547
2.29 2.17
am + tr ca am
8.80 10.5 10.2 5.25
0.322 0.419 0.387 0.253
3.38 2.18 2.08 4.22
ap (I+) + ca am + tr ca
9.84 4.59 4.57
0.438 0.215 0.226
2.06 5.86 3.57
ap cl+) SP (I+)
ca
-
110
110
1.00
110
1.33
83
l.67d
66
nP (I+)
ap (1-t)
+ ca
aP (lf)
2.OOd
55
2.33d
47
7.46 8.60
0.220 0.290
2.49 2.09
ap @+I + ca + ar al, (2-t)
3.33d
33
4.30
0.200
2.29
“P (3+)
0
cc
0.30
0
0.033
3300
0.51
tr
0.167 0.333 0.667 1.00
660 330 165 110
5.0
0.167 0.333 0.667 1.00
None in 2 expts.
5.0
1.33
None in 2 expts.
1.33
83 4.60
1.16
5.0
1.67
4.7
1.50
1.67
m >lOO
2.00
+ tr ca
ca + tr arf ea
aP (2+)
None in 2 expts.
66
130
+ ca + ca
1.62
ap (a+)
MM
TABLE Initial composition of solutions”
III-Continued
Fina! composition of solutions ._.~
Ca (IIlmM)
C (mW
&I
2.00 5.0
C/P
55
110
X-ray patternC
Ca/P (molar!
(S,
(ZiR,
2.90 2.u 2.52
0.680 0.800 0.607
2.27 2.04
2.15
2.19 1.88 1.78
1.96 “P
2.33
47
2.07 2.11
0.720 0.690 0.797
3.33
33
0.98
1.05
1.93
4.m
27.5
0.485
1.39
1.73
5.00
22
0.403
2.34
1.65
0
02
0.26 0.30 0.35
0
1.67 3.33
60
5.00 6.67
1.5
110
8.33
10.0
33 22 16.5
13.4
11
1.0
8.3
aP (3f)
ca
1.67 1.5
3.33 5.w 6.Gi
None in 3 expts.
None in 1 expt.
1.50
a.33
1.16
7.55 7.60 7.42 7.35
1.66 1.78
8.85 8.45
1.65 1.79
9.52
1.81
1.26 0.29 0.71 <0.25 <0.25 0.82
13.33
cc
@+I
<0.25 0.26
11.7 10.8
1.68
1.70
1.57
1.69
<0.25
14.9
1.65
CO.25 <0.25
14.7 14.5
1.71 1.71)
16.67
6.6
0 5.0 6.67 8.33 10.00
m 22 16.5 13.4
13.33
8.3
0.57
12.3
1.62
16.67
6.6
0.363
15.8
1.62
20.00
5.5
19.0
1.41
1.0
I1
ap (3+)
0 5.0 6.67 8.33
None in 2 expts.
10.00
110
<0.25
aP
(3+)
a See Table I for the concentrations of the other ions. Each line in the Table represents the results of one individual experiment, unless stated otherwise. The solutions were kept at 37’ C., usually for 72 hr., and the initial PH was 7.30 f 0.05. Some experiments were terminated after shorter periods of time (see -0). The final pH had dropped at most a few tenths of a pI1 unit. b-dSee the corresponding legends of Tahle IT. * Experiment terminated after 5 hr. f Experiment terminat.ed after 24 hr. a Experiment terminated after 72 hr. 131
132
HACHRA,
TRAHTZ,
3 days. Less precipitate could form at the high bicarbonate level because lower calcium concentrations were employed. Also, the solutions had a larger buffer capacity. For these reasons, the pH of these solutions dropped at most a few tenths of a pH unit during the precipitation. The precipitation of calcium carbonate was also prevented by trace amounts of pyrophosphate. As little as 4 &’ of sodium pyrophosphate (about 1 ppm) prevented such precipitation at a calcium concentration of 75 mM and a bicarbonate level of 22 mM.
AND
SIMON
The calcium carbonate precipitates formed in the absence of phosphate and kept in contact with the supernatant solution for 3 days consisted of calcite, often mixed with a small amount of aragonite. Examination of the earliest precipitates revealed the presence of considerable amounts of aragonite beside the calcite. Rut during the 3 days contact with the supernatant the aragonite transformed completely or almost completely into calcite. Figure 1 demonstrates such a transformation in the precipitate from a solution containing 12.5 mM calcium, 110 mM bicarbonate, and 0 mM
FIG. 1. Transformation of aragonite into calcite. X-ray patterns of: (a) halibut otolith (pure aragonite) and of precipitates isolated after: (b) 5 hr. (calcite + much aragonite), (c) 24 hr. (calcite + some aragonite), (d) 72 hr. (calcite + trace of aragonite) from a solution containing: 12.5 mn/r Ca, 110 mM bicarbonate and 0 mM phosphate, at p = 0.26,37” C., and pH 7.30 + 0.05.
PRECIPITATION
OF CALCIUM
CARBONATE8
AND PHOSPHATES
133
FIG. 2. X-ray patterns of calcium phosphate precipitates, illustrating the increase in their cystallinity with decreasing C/P ratio of the solution :12.5 mM calcium, 110 mM bicarbonate; see Table III. (a) Amorphous calcium carbonate phosphate (C/P = 83); (b) extremely poorly cystallized apatite + well-crystallized calcite (C/l’ = 66); (c) very poorly crystallized apatite (C/P = 47). phosphate (see Table III). In the presence of phosphate, aragonite did not form at all or only in trace amounts, even in the earliest precipitate. On the other hand, pure aragonite powder, obtained from a halibut otolith, did not transform into calcite when kept in contact with solutions from which no spontaneous precipitation of calcium carbonate or phosphate occurred. These findings indicate that aragonite transformed into calcite only when it was kept in the solutions together with calcite. By raising the phosphate concentration sufficiently, precipitates could again be obtained. These consisted of either amorphous calcium carbonate phosphate or of more or less poorly crystallized apatite., Amorphous precipitates formed at the bicarbonate level of 22 mM, when the C/P ratio was at least 44 and the calcium concentration was > 25 mM. The required minimum C/P ratio and calcium concentra-
tion were 66 and 5 mM, respectively, at the bicarbonate level of 110 mM. The crystallinity of the apatitic precipitates increased with decreasing C/P ratio (see Fig. 2) and decreasing calcium concentration. In many of the precipitates larger or smaller amounts of calcite and, occasionally, traces of aragonite were found beside the amorphous phase or the apatite. Such crystallized calcium carbonates appeared when the phosphate concentration had diminished considerably during the precipitation. 11ISCUSSION
THE EFFECT OF PHOSPHATE ON THE PRECIPITATION OF CALCIUM CARBOKATE The results of our experiments show that, under physiological conditions, the spontaneous precipitation of calcium carbonate
13-l
BACHRA,
TRAUTZ,
can be prevented by the presence of low concentrations of phosphate ions. At both bicarbonat,e levels such precipitation was prevented when the phosphate/bicarbonate concentration ratio was higher than about l/300. Above this ratio, the competition of the phosphate ions with the carbonate ions for the calcium ions was evidently sufficient to inhibit the nucleation and crystal growth of calcite and aragonite, while the phosphate concentration was still too low for the nucleation of calcium phosphate. Pyrophosphate and hexametaphosphate inhibit the precipitation of calcium carbonate in a similar way (16-19). Table IV shows the range of concentrations of some of the most import#ant ions present in a number of biological fluids, as reported in the literature. Our findings indicate that calcium carbonate deposition should not take place in these fluids on account of the inhibitory effect of the phosphate ions. Even when solutions similar in composition to these biological fluids were seeded with calcite, no precipitation of additional calcite was obtained (20). The composition of the endolymph in the
COMPOSITION
OF SOME BIOLOGICAL
mM
Na
Cl
ANI)
human inner ear, as reported in the literature (see Table IV), is similar to that of serum ultrafiltrate. This suggests that the human statoconia, which consist of calcite, are deposited in a fluid which differs in composition from endolymph. Our chemical analysis of a halibut otolith, which gave the X-ray patt,ern of pure aragonite, showed no detectable phosphate. The phosphate ions would have been coprecipitated with the calcium carbonate, had any been present in the fluid at the site of otolith formation (see Tables II and III), since the phosphate ions are bound more strongly to the calcium ions than are the carbonate ions. We postulate therefore that such otoliths are formed in a solution essentially free of phosphate ions. A report in the literature (21) states that calcite calculi have been found in the pancreatic tissue of a patient suffering from chronic pancreatitis. In this connection, it is of interest that pancreatic juice contains bicarbonate in large concentrations and can be very low in phosphate. Calcium carbonate has not been found in urinary calculi (22, 23). This is most likely
TABLE IV FLUIDS WITH RESPECT K
c8.a
SIMON
TO THE MOST
IMPORTANT
Pb
CC
Msa
INORGANIC
IONS
PH
Reid
_-
mammalian serum
136-171
90-156
3.4-10.2
1.8-5.0
0.5-1.8
0.7-3.0
-
4.6-6.4
3.3-5.9 -__
0.45-2.55
-
102-160
2.4-7.9 ~---
2.04.1
0.58-0.99 --__ 0.5-7.9
0.7-5.8
0.08-0.53 --__
2.44.8 __------__-_-_
2-48 ~--~__-__ 0.57-3.2
0.15
0.008&0.41
1630
7.3-7.4
46b
-----------__ bird
serum ----__-__--
reptilian --
serum
loo-186 -----__
human saliva _~____
3.5-57.98.4-17.712.7-37.80.9-2.4 ~---__ --------
human pancrea113-153 tic juice ---~-~---____~human lymph ---__ crustacean olymph
endo-
6-30
54-95
2.6-7.4
1.1-1.6
loo-123
128-169
0.7-1.45
56-590
180-1690
0.546
46c -___
-
46d
5.6-7.6
46e
66-127
8.6-8.8
46f
7.7-14.5
-
~_-------__--__-_
-----------
hem-
-__-
1.2-1.3
0.30.7
47, 48
-~------------__-__-______-_ 5.342.5
2.5-53
0.164.48
5.5-32.57.5-8.5
0 Only part of the calcium and magnesium is present in ionic form. b Phosphorus present as inorganic phosphate. c Bicarbonate. d The concentration ranges of the ions were derived from the values tabulated
in the cited
29b,46a, and 49
references.
PRECIPITATIOX
OF CALCIUM
CARBONATES
AND
PHOSPHATES
135
ways to deposit fine-grained calcite in COIlagenous or chitinous fibrous matrix but were not successful in obtaining such deposits (31). This indicates that calcite deposition in the tissues of these arthropods occurs under conditions which we have not yet been able to duplicate in the laboratory. The simultaneous growth of a large number of nuclei in a small volume of tissue may give rise to a multitude of very small crystallites. The smallness of such crystallites may be due in part also to impurities, like phosphate and pyrophosphate, which at higher concentrations, inhibit the precipitation of calcium carbonate, and at lower coneentrations disturb its crystallization to such an extent that the growth of the crystallites into larger crystals is prevented (16-19). In our experiments, however, we never observed such fine-grained deposits, even when phosphate or pyrophosphate was present. Such deposits may have escaped our attention, because the bulk of the phosphate and pyrophosphate is removed from the solution with the precipitate at an early stage of the precipitation. The large quantities of wellcrystallized material formed after the removal of the phosphate or pyrophosphate may have masked the fine-grained fraction on the X-ray patterns. Biological deposits of pure aragonite are found in the otoliths of bone fishes (6a, 7-10) and in the skeletons of many invertebrates (6b). The results of our experiments indicate that deposits of calcite-free aragonite do not easily transform into calcite under physiological conditions. Yo adequate explanation, however, is available at present for the frequent biological deposition of metastable aragonite in preference to the stable calcite, though it is known that such deposition is SPECIAL FEATURES 0~ SOME BIOLOGIC~~L facilitated by elevated temperature (32-34), CALCIUM CARBONATES by certain metal ions (35), and by epitactic fact’ors (36). That epitactic factors can cause The calcite and aragonite precipitates the deposition of metastable instead of the obtained in our experiments were always well crystallized and coarse-grained, as stable modification has been shown also shown by t.he spotty appearance of the with other salts (37, 38). A third modification of calcium carbonate, X-ray reflections. We never obtained crysvaterite, has been demonstrated in the tallites as small as the very fine-grained calcite deposited in vivo in the carapace and calcified tissues of certain invertebrates tendon plates of certain arthropods (1~). (6b). The factors causing its deposition in the tissues are at present unknown. In other studies we att,empted in various
due to the high phosphate concentration in this fluid. The composition of human saliva precludes the crystallization of calcium carbonate in salivary and dental calculi (2426). The presence of calcite in dental calculus from the horse, dog, ferret, and hamster (27) makes it of interest to study the composition of the saliva in these animals. In some crustaceans the hemolymph has a composition similar to that of our experimental solutions from which amorphous calcium carbonate phosphate precipitated. Small amounts of such amorphous deposits have, indeed, been found in the skeletons of some crustaceans (lb, lc, 2). The principal mineral deposit in these tissues, however, consists of calcite. One would not expect such deposits to form in the hemolymph, since its phosphate concentration is too high to allow this. On the basis of the above considerations we feel that cellular activity may be required to modify the composition of the tissue fluid at the sites of calcium carbonate deposition in most tissues where such deposits occur. For instance, the cells could provide a raised carbonate level. Large amounts of COZ may be formed by metabolic processes and be rapidly hydrated under the influence of carbonic anhydrase. This enzyme was recently demonstrated in exceptionally large quantities in the inner ear of the cat (28). It also occurs in high concentrations in the oviducts of birds (llb) and in the calcium carbonate depositing tissues of many invertebrates (29a, 30). The phosphate concentration may be lowered at the sites of calcium carbonate deposition by metabolic processes favoring the formation of phosphate esters.
136
BACHRA.
TRAUTZ,
THE EFFECT OF CARBONATE ON THE PRECIPITATION OF CALCIUM PHOSPHATE
AND
SIMON
of such amorphous precipitates is also governed by some sort of an ion product relationship. The final Ca and P concentrations in the In earlier experiments (2) it was found solution after 3 days (Tables II and III) that the presence of carbonate in the soluwere higher when the precipitate was amortion reduced the crystallinity of the precipitated apatite. These experiments were made phous than when it was apat,itic. They also were higher in the experiments at the high under conditions favoring rapid spontaneous than at the low bicarbonate level. This indiprecipitation at concentrations of calcium, cates t,hat the presence of bicarbonate inphosphate, and bicarbonate considerably above the physiological range. An amor- creases the solubility of the solid phase and is more phous calcium carbonate phosphate was that an amorphous precipitate soluble than an apatitic one. It must be obtained when the C/P molar ratio in the borne in mind, however, that the precipisolution was 17 or higher. The precipitate tated solids and the supernatant solutions gradually changed into a poorly crystallized were usually not yet in equilibrium after apatite when it was kept in contact with its 3 days. During the deposition of larger supernatant solution for several days. In the present series of experiments condi- amounts of precipitate the composition of the solution changes markedly and with it tions were employed favoring a slow precipitation at physiological concent,rations and also the composition and character of the precipitating solid. For instance, calcite pH. The amorphous precipitates obtained under these conditions were found to be frequently crystallized after the amorphous stable when kept in contact with the super- or apatitic precipitate formed earlier had removed enough phosphate from the solunatant solution for 3 days, but the minimum tion to raise the C/P ratio sufficiently. Also, C/P ratio required was considerably higher: when an amorphous precipitate is depositing, 44 at 22 mM bicarbonate, and 66 at 110 mM bicarbonate. Evidently, the carbonate ions the calcium concentration in the solution compete with the phosphate ions for the will decrease and this may enable the available calcium on the surface of the solid deposition of an apatitic precipitate at a later stage (20). phase and, at a sufficiently high C/P ratio, The chemical analysis of the amorphous can prevent the orderly crystallization of the apatite. Such competition is more effec- calcium carbonate phosphat,es, which contive when the precipitation is rapid and a tained no or only a trace of calcite according lower C/P ratio suffices for the formation of to the X-ray pattern, showed Ca/P molar an amorphous precipitate. ratios of about 2.1-2.3, while the theoretical As the phosphate is bound more strongly value for apatite is 1.67. The apatitic preto the calcium than is the carbonate, a great cipitates had Ca/P ratios ranging from 2.2 excess of carbonate in the solution is re- to 1.4. Apatitic precipitates with a high quired to prevent the crystallization of apa- Ca/P ratio were usually obtained from solutite and to cause the formation of an amor- tions with a high C/P ratio. They were phous precipitate. For the same reason, the poorly crystallized and probably conbained C/P ratio in the amorphous precipitate is also a large amount of an amorphous phase alwa,ys many times (1 to 2 orders of magnitude) lower than the C/P ratio in the solu- (20). The higher Ca/P ratios of the amorphous precipitates indicate that, at least, tion from which it was formed (Id). At a particular C/P ratio, the lower P 20-27s of the Ca ions in such material are (and C) concentrations required higher Ca bound to carbonate. The actual percentage should even be higher than that, since conconcentrations for the spontaneous precipisiderable amounts of HP04 and Na ions are tation of an amorphous calcium carbonate phosphate than the higher P (and C) con- also present, which tend to lower the Ca/P ratio of the precipitates. centrations. This suggests that the formation
PRECIPITATION
OF CALCIUM
CARBONATES
THE EFFECT OF T~VPURITIES ON THE CRYSTALLINITY OF THE BIOLOGICAL APAT~TES
The amorphous precipitates of calcium carbonate phosphate found in the carapace and tendon plates of certain arthropods and in the concretions of tapeworms (lb, lc, 2, 12) may form under condit,ions similar to those employed in our experiments. The Ca/P ratios in the tapeworm concretions are higher than 3 (12). We feel that these biological depo,sits, in spite of their high Ca/P ratios, are completely amorphous on account of the presence of magnesium. Experiments to be reported (20) show that the presence of magnesium ions facilitates the formation of amorphous calcium carbonate phosphate and slows down or prevents its crystallization into apatite. The apatite crystallites of calcified tissues are very small. In bone (39, 40), dentin (41, 42), and calcified cartilage (43), platelets and needles have been found with dimensions varying between less th$n 100 and somew.hat more than 1000 A. Our results suggest that the smallness of these crystallites is due to the presence of impurities, like HPO,, carbonate and magnesium ions, which cause lattice distortion and limit the crystal size. The apatite crystals in dental enamel (le, 44, 45) are larger and better crystallized than those in t,he ot.her mineralized tissues. They contain less of the various impurities (Mg, Ka, COa, HPO+ citrate, etc.). It is likely therefore that the ameloblasts modify the tissue fluid in which these crystals form, so that it contains less of these impurities than the tissue fluid in bone and dentin. ACKNOWLEDGMENT We acknowledge the assistance of Mr. Conetta in the early phases of this work.
A. R.
REFERENCES 1. TRAUTZ, 0. R., (a) Ann. A:. Y. Acad. Sci. 60, 696 (1955); (b) ibid. 86, 145 (1960); (c) Progress Reports Contract DA-49.007.MD-765, Research & Development Division, Office of the Surgeon General, U. S. Army, 1957, 1958 and 1959; (d) unpublished results, (e) Ann. Dent. 12, 47 (1953).
AND
PHOSPHATES
137
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