- Email: [email protected]
The removal of Sr85 and Ca45 from bone in vitro
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
The Removal JOSEPH From the Veterans
lo.?,
168-174 (1963)
of Srs and Ca45 from SAMACHSON
Administration
Hospital,
AND
Hines,
Received
March
Bone in Vitro’
HILDA Illinois;
LEDERER
and Montefiore
Hospital,
New York
21, 1963
Defatted bone powder, anorganic bone, ashed bone, and apatite which had taken up Ca45 and Sr*6 under different conditions were subjected to wash solutions after standing for different lengths of time or after being subjected to heat treatment. A number of factors affected the ratio of Cad5 removed to SF removed, but under a given set of conditions a change in the ratio could be used as a criterion of recrystallization. The decrease in amounts removable after different periods of standing could be ascribed primarily to processes occurring in the organic matter of bone, and to a lesser extent in the hydration layer and on the crystal surface. There was no evidence of genuine recrystallization at room temperature.
The retention of radioisotopes is a problem which has become of increasing concern in recent years and has aroused the interest of many investigators. Radioactive isotopes of strontium and calcium are taken up by the skeleton, at first mostly by exchange with the calcium of already formed bone, and later on chiefly by the accretion of new bone (1, 2)) becoming increasingly difficult to remove as the length of time after administration increases (3,4). After accretion, such potentially dangerous isotopes as SrgO are firmly fixed in the hard tissues and can be removed only by resorption during the normal processes of bone remodeling, or by the dissolution of bone with such agents as ammonium chloride (4) or parathyroid hormone (5). Although Srs5 and Ca45 are relatively easy to remove before accretion has taken place, even exchange may not be completely reversible. Exchange with the calcium of the bone surface must be preceded by the diffusion of calcium or strontium through the organic matrix of bone (6,7) and through a hydration layer of the hydroxyapatite crystals (8), and it may be followed by 1 This National Diseases,
incorporation of the radioisotopes into the interior of the crystals. The physical chemical processes involved are complicated, occurring to a large extent simultaneously with each other and with the physiological processes of accretion and resorption, and they are intimately connected with the still uncertain mechanisms of bone formation. The removal of radioisotopes from bone requires a reversal of these processes. Dallemagne and his co-workers (9, 10) have used Ca45 to study recrystallization and throw light on the nature of bone and bone mineral. In the present study, Sr85 has also been utilized, not only to supply additional data, but because the Ca45/Sr85 ratio of uptake or release under different conditions provides information that the values for individual radioisotopes do not. The effects of time and temperature on bone which had taken up Ca45 and SF in vitro under different conditions prior to the removal of the radioisotopes have been investigated in the hope of distinguishing between diffusion through the organic matrix, diffusion through the hydration layer, and recrystallization. EXPERIMENTAL
work was supported by grant A-5572, Institute of Arthritis and Metabolic National Institutes of Health.
Bone was prepared from canine tibiae which were dried, ground to between 60 and 100 mesh, 168
REMOVAL
OF SI?J AND
Ca46 FROM
TABLE
BONE
IN VITRO
169
I
EFFECT OFTIME ON REMOVAL OF Ca4” AND Sr*6~~~~ BONE POWDERBY BUFFER SOLUTION Carrier in original radioisotope solution None None Ca Ca Sr Sr + Ca Sr + Ca Ca + P Ca + P Sr + P Averages
% of bone radioisotope removed by buffer Immediately SP Ca45/Sr”6 Ca4” 25.4 28.5 32.6 25.5 31.3 35.4 36.9 38.0 38.0 38.2 32.98
34.8 38.4 42.0 32.7 46.0 45.3 48.2 44.8 45.5 44.2 42.19
0.73 0.74 0.78 0.78 0.68 0.78 0.77 0.85 0.84 0.86 0.781
After 72 hr. SP Ca43/Sr85 C&s
Cd6 13.2 13.6 14.7 12.5 14.0 14.3 10.2 11.4 22.4 14.03
defatted, and dried again at 105°C. To obtain “anorganic” bone, part of the crushed bone was refluxed with ethylenediamine, washed with methyl alcohol, and dried. Ashed bone was obtained by heating crushed bone for about 2 hr. at 550°C. For purposes of comparison with ashed bone, part of a single apatite crystal2 was crushed and ground to below 200 mesh. SrssC12 without carrier and Ca4%12 (specific activity of approximately 20-30 PC. per ml.) were used. Solutions buffered with 0.04 X sodium Verona1 and hydrochloric acid to a pH of approximately 7.3 were prepared containing Sr*S and Ca45, either without added carrier, with CaC12, or SrCls as carrier, or with both. In shaking with bone powder and ethylenediamine-treated bone, carriers were used at a concentration of 2.5 mM; in shaking with apatite and ashed tibia, at 1.25 mM. In some cases, sodium phosphates were added to give a final concentration of 3 mg% P. One hundred-mg. samples of ground bone, ashed bone, or apatite were “anorganic bone,” treated at 37°C. for 2 hr. with 50 ml. of solution in flasks which were’ attached to a mechanical stirrer and rotated to give the effect of gentle shaking. The sample was then removed and washed by shaking with fresh W-ml. portions of solution, usually containing buffer alone at a pH of 7.3. The effect of washing with water was compared with that of washing with 2.5 mM CaClg solution in a 2 x 2 x 2 factorial experiment, in which the other factors were bone powder vs. “anorganic” 2 This apatite had a high fluorine content and was obtained through the courtesy of Clifford Frondel, Professor of Mineralogy, Harvard University.
20.0 20.7 22.3 17.4 15.2 18.6 15.4 13.3 26.4 18.81
0.66 0.66 0.66 0.72 0.92 0.77 0.66 0.86 0.85 0.751
5.9 8.1 7.9 10.3 10.9 12.6 11.0 16.4 10.39
After 192hr.
se
Ca46/S19"
10.4 15.2 20.0
0.57 0.53 0.40
13.8 13.8 14.9 12.3 20.9 15.16
0.75 0.79 0.85 0.89 0.78 0.695
bone powder, and uptake of radioisotopes in the presence of 2.5 mM CaCl* carrier or its absence. In some experiments, the bone samples, after taking up radioisotopes, were heated at approximately 475” or 525°C. for varying lengths of time, while in others they were allowed to stand without drying for 0, 72, or 196 hr. They were then shaken with the buffered wash solutions indicated above, and these were analyzed for Ca4S and Srss. The original solutions were also analyzed, after shaking with bone samples, for P, and if no stable Sr was present, for stable Ca. Bone samples were ashed, unless organic matter had been removed from them, dissolved in HCI, and also analyzed for Ca and P, as well as for Ca4h and SrsS. Experiments were usually performed in duplicate. Srs5 was determined in a scintillation counter of the well type, Ca45 by precipitation with stable Ca, as previously reported (II), counting being done in a thin-window gas flow counter. Phosphorus was determined by the method of Fiske and SubbaRow (12), stable calcium by precipitation with ammonium oxalate, and titration of the dissolved precipitate with potassium permanganate. RESULTS
Table I shows the effect of time on the removal of Ca45 and SP taken up by bone from various solutions. From 25 to 38% of the Ca45 and 33 to 48% of the Srs5 could be washed out by buffer solution used immediately. After 72 hr., the average percentage of removable radioisotope had decreased by more than half. After 120
170
SAMACHSON
AND TABLE
EFFECT OF TIME
II
ON REMOVAL OF Cad5 AND Sr85 FROM ANORGANIC BONE BY BUFFER SOLUTIONS ‘% bone radioisotope removed by buffer
Carrier in original radioisotope solution
After 72 hr.
Immediately C@
srss
None Ca Ca Ca + Sr Ca + Sr Sr Sr
16.2 18.4 18.7 19.6 17.9 17.6 16.9 16.7
22.2 25.1 26.6 27.3 26.6 26.3 28.6 27.5
0.73 0.73 0.70 0.72 0.67 0.67 0.59 0.61
13.0 12.3 13.3 12.9 14.3 12.5 14.1 11.5
Ca + P Ca + P Sr + P Sr + P Averages
15.9 14.9 20.2 18.4 17.62
21.7 21.5 29.1 26.9 25.78
0.73 0.69 0.69 0.68 0.684
9.9 9.9 12.3 11.0 12.25
None
CaJ5jSrs~
CC&
TABLE III UPTAKE OF RADIOISOTOPES BY BONE POWDER AND BY ANORGANIC BONE ‘% of radioisoto~;ae&riginal
solution
Carrier C&4’
Bone powder None (6)= Ca (4) Sr (2) Sr + Ca (6) Ca + P (6) Sr + P (3) Anorganic bone None (6) Ca (6) Ca + Sr (4) Sr (4) Ca + P (6) Sr + P (6) a Number
LEDERER
Sr86
CaWSlg~
23.5-25.618.4-21.1 7.6-15.0 6.0-12.1 19.8-20.0 8.7, 9.2 8.2-10.0 5.3- 6.2 15.8-17.912.0-13.0 20.4-21.4 9.1- 9.5
1.20-1.32 1.24-1.40 2.17,2.28 1.5c1.72 1.31-1.43 2.15-2.35
49.5-64.241.4-55.9 29.5-35.020.9-27.2 23.7-26.212.2-13.9 40.3-43.818.0-21.7 33.G48.425.3-38.2 47.&50.621.6-24.5
1.15-1.20 1.29-1.36 1.88-1.94 2.02-2.24 1.26-1.35 1.99-2.21
of replicates
in parentheses.
additional hr., there was a further highly significant decrease of almost 4% in removable Ca45 and SF. The ratio of Ca45/ Srs5 did not change significantly with time. More Ca45 could be removed from the samples which had been shaken with phosphate than from those not shaken with phosphate, the difference being significant at the 5 % level. There was no significant
After 192 hrs.
.caqws
C@
SC
Ca”5jSF
18.9 17.3 18.2 17.3 24.6 20.1 23.3 20.1
0.69 0.71 0.73 0.75 0.58 0.62 0.61 0.57
12.0 13.1 11.5 12.8 -
16.1 16.7 15.9 17.7 -
0.75 0.78 0.72 0.72 -
14.4 14.7 21.7 19.9 19.21
0.69 0.67 0.57 0.55 0.645
10.1 10.0 13.6 14.6 12.21
14.1 13.8 21.3 22.1 17.21
0.72 0.72 0.64 0.66 0.714
Sldb
difference in Srsj washed out, but the ratio of Ca45/SF was significantly higher at the 1% level after the use of phosphate, both for samples washed immediately and for all samples washed. Results for anorganic bone are presented in Table II. Smaller fractions of the Ca4j and Sr*5 taken up were removed by immediate washing. However, as anorganic bone took up more than twice as much radioisotope as untreated bone (Table III), the amounts removed, expressed as fractions of the radioisotope originally available for uptake by equivalent amounts of bone salt, were about equal. At 72 hr., the average amounts removed had decreased by about a third. An analysis of variance showed that the effects of both time and treatment were highly significant, the significance of treatment being due chiefly to the calcium carrier plus phosphate. In contrast to what is seen in Table I, the amounts washed out after this treatment were significantly lower. There was no further decrease of removable radioisotope at 192 hr. The ratio of removable Ca4j/SrS5 depended on pretreatment. It appeared to decrease during the first 72 hr. and then rise again. The differences were small and probably not significant, although the unusually small variability of duplicate samples made them appear significant.
REMOVAL
OF SP
AND
Ca46 FROM
TABLE EFFECT
OF TIME
ON REMOVAL
BONE
IN
171
VZ’Z’RO
IV
OF Ca46 AND Sra5 FROM AMIED
TIBIA
BY BUFFER
SOLUTION
Y0 bone radioisotope removed by buffer Carrier in original radioisotope solution
None (2)a Ca (2) Sr (2, I)* Ca + Sr (4) Averages (10, 12)c
29.7 35.7 45.2 34.2 35.73
a Number of replicates * Immediate treatment c Averages of individual
After 72 hrs.
Immediately
in parentheses. in duplicate; after values.
39.2 44.2 52.4 35.5 41.33
0.76 0.81 0.87 0.97 0.874
35.5 29.1 32.1 29.7 31.33
42.3 34.8 40.9 32.3 37.23
0.84 0.84 0.79 0.95 0.855
72 hr. in quadruplicate.
possibly because error was greater when small amounts were involved. The data for apatite are similar and are therefore not listed in detail. There was even W~/S+ taken Cd6/Srss in Carrier in original up (% original wash solution less uptake and more variability, and again solution (% in bone) solution) no change with time. The average Ca45/Sr85 ratio in the wash solutions was greater than None (4)5 1.30 0.89 1. Table V shows, however, that these Ca (4) 1.13 0.99 Sr (4) 1.51 1.25 ratios were less than the corresponding Ca + Sr (12) 1.12 1.03 Ca45/Sr85 ratios for bone uptake, i.e., the apatite continued to show some preferential a Xumber of replicates in parentheses. retention of Ca45. The data of Table VI indicate that wash stable Ca removed The uptake of radioisotope by bone from solutions containing the original solution equalled the sum of about twice as much Ca45 and SrE5 as solubuffer alone. Again a radioisotopes found in bone and in the wash tions containing was solution, and the values listed for Ca45, larger fraction of the radioisotope SF, and their ratios in Table III agree removed from untreated than from anorganic bone. The ratio of Ca4j/Srs5 was very with those found when no wash solution was used and the bone was analyzed for significantly greater when calcium was used in the wash water and was also greater for total uptake (13). As the number of replicates varied from 2 to 6, ranges are given untreated than for anorganic bone. Amounts instead of standard deviation, although the washed out were the same whether the latter can easily be estimated from the uptake of radioisotope had been from solutions containing buffer alone or buffer plus former (14). Ashed bone took up smaller amounts of calcium. The fraction of Ca45 and SP removable radioisotope from the original solution, calcium averaging 25 % Ca45 and 20 % SP in the with wash solutions containing absence of carrier, and 7.2 % Ca45 and 4.3 % decreased when the bone had been heated (Table VII). On one sample of bone heated Sr85 in the presence of calcium plus stronat 475°C. for 2 hr. the effect was minor, tium. About a third of the Ca45and slightly possibly because of failure to attain the more of the SP could be washed out (Table temperature soon enough. The other data IV). There was no significant change with time in Ca4j, Sr85, or Ca45/Sr85 ratios, the indicate no difference in amounts removable latter being higher than in the case of whether time of heating was 2, 6, or 24 hr. An increase in temperature to 525°C. untreated or anorganic bone. Variability was greater than in the previous cases, reduced the amount removable by half. TABLE
RATIOS
V
OF Ca4s/Sr85 TAKEN UP BY APATITE WASHED OUT BY BUFFER
AND
172
SAMACHSON
AND TABLE
EFFECT
OF CONDITIONS
of radioisotopes
Washed without
Ca
Ca
Anorganic bone Washed without Washed with
Ca
Ca
Removal Powdered bone Washed without Washed with
with
TABLE
475°C.
24 hr.
525°C.
2 hr. 6 hr. 24 hr.
Ca
Shaken with
Ca
Ca45 14.5 12.2 12.2 12.1
Srs5 10.1 9.5 9.2 9.3
Ca46/SrsS 1.44 1.28 1.33 1.30
63.5 61.6 56.5 54.0
51.5 49.6 48.9 49.1
1.23 1.24 1.16 1.10
39.2 39.2 37.9 36.7
27.7 28.3 27.1 26.4
1.42 1.39 1.40 1.39
from bone,
70 in bone
24.3 23.1 49.3 45.5
30.8 29.0 55.2 50.2
0.79 0.80 0.89 0.91
26.9 27.9 51.6 47.1
33.7 31.6 51.1 48.4
0.80 0.88 1.01 0.97
Ca
14.8 15.7 39.1 35.0
21.0 22.0 45.0 41.1
0.70 0.71 0.87 0.85
14.8 15.3 34.8 36.0
21.3 21.9 42.1 44.7
0.69 0.70 0.83 0.81
Ca
VII
23.08 20.83 20.14
6 hr.
solution
Ca45/SrsS 1.27 1.31 1.28 1.24
y. removed
2 hr.
ON
Ca
Cd5
None
SOLUTION
Srs5 26.3 25.5 23.9 25.1
EFFECT OF HEATING OF BONE ON SUBSEQUENT REMOVAL OF SrsS AND Ca45 BY WASHING Heating
70 in original
IN WASH
Ca4& 33.3 33.3 30.6 31.2
of radioisotopes
Ca
Anorganic bone Washed without Washed
by bone,
Shaken without
bone
Washed with
VI
OF UPTAKE AND PRESENCE OF CARRIER REMOVAL OF Ca45 AND Srs5 FROM BONE
Uptake Powdered
LEDERER
5.89 17.34 6.07 6.08 5.63 5.81
2.89 2.74 2.98
sr=
Ca46/Sr85
27.36 22.09 21 .OY
0.84 0.91 0.95
Average
0.90
10.40 24.05 12.17 12.32 11.82 11.78
0.57 0.72 0.50 0.49 0.48 0.49
Average
0.54
5.78 4.21 5.12
0.50 0.65 0.58
Average
0.58
Again with the indicated exception, the ratio of Ca45/Sr85 removed was about 0.5, the same at both temperatures and various times of heating, and very significantly different from the value of 0.9 before heating. DISCUSSION
-
A comparison of the considerable effect of standing for 72 hr. on powdered whole bone (Table I) with the smaller effect on bone which lacks organic matter (Table II) indicates that processes occurring in the organic matrix are of major importance in determining the extent to which radioisotopes can be removed. During these 72 hr. the radioisotopes either diffuse through the organic matrix to the crystals (6, 7) or are more firmly bound by the organic matrix itself. The former hypothesis appears more likely, but the latter cannot be completely excluded. Previous data obtained on the uptake of radioisotopes by organic matter left when bone is decalcified with ethylenediaminetetraacetic acid (13) have
REMOVAL
OF Sr*s AND
C a45 FROM
been used to calculate the amounts of Ca45 and Sr86 taken up by organic matter in the present study. Although these amounts are too small to account for the changes that take place in 72 hr. in Table I, the original untreated organic matter may have a greater capacity to bind these elements, and the organic-inorganic interface may also be involved. Dallemagne has repeatedly emphasized (6, 7) that the mineral in the organic bone differs from the isolated bone mineral, the organic matter apparently stabilizing the mineral structure. Conversely, the mineral may stabilize the organic structure and permit greater uptake of radioisotopes. A comparison of the effect of standing on ethylenediamine-treated bone (Table II) with the lack of effect on ashed bone (Table IV) indicates that the hydration layer also takes part in controlling the removal of radioisotopes, although to a lesser extent than the organic matrix. This layer serves as a medium for the dissolution and redeposition of calcium and phosphate ions from the crystal surface and for the exchange of ions which enter from the bulk solution (8). Kot only calcium and strontium but sodium, magnesium, and hydronium ions of bone may exchange with calcium or strontium from the solution and defects in the crystal surface may be created or eliminated. Although these processes affect the amount of radioisotope removable, and may be considered a form of recrystallization, they differ considerably from the recrystallization which involves penetration into the crystal interior. They differ also from the recrystallization which Arnold and Jee (3) reported to take place in viva. The latter involves the physiological dissolution of bone and the formation of new bone. The ratio of removable Ca45 to Srs5 depended on the nature of both the solutions from which the radioisotopes were taken up and the bone powder. The changes in amounts washed out after uptake from solutions containing Ca and P is of special interest. In this case, the amounts removable from powdered bone as well as the ratio of removable Ca45 to Srs5 were greater than after uptake from buffered solutions. On
BONE
IN VITRO
173
the other hand, the amounts removable from anorganic bone were lower after uptake from Ca and P solutions than after uptake from buffered solutions. The use of Ca and P in the amounts indicated probably led to the deposition of calcium phosphate, In whole bone this may have been in the form of colloidal tiny particles which deposited in the organic matrix and were easily dissolved again, while in anorganic bone, new layers may have been added to already existing apatite crystals and may have been more difficult to remove. The recrystallization of bone and bone mineral has been studied by a number of investigators, including Arnold and Jee (3), who washed bone with distilled water and calcium acetate solutions, and Dallemagne and his coworkers (6, 7, 9), who carried out numerous experiments with hydrochloric acid. Dallemagne used as his criterion of recrystallization the pattern of removal with successive washes of acid, assuming that these would more or less regularly attack successive layers of bone salt crystal. Both investigators found that removal of the radioisotope became more difficult as time after deposition increased, and concluded that recrystallization was taking place. Difficulty of removal alone, however, is not a sufficient criterion of recrystallization, and another one was sought for this study, based on Dallemagne’s observation that heating at 400°C. for 24 hr. was sufficient for Ca45to penetrate uniformly into the interior of bone salt crystals (15). It was expected that Sr85, because of its larger radius, would penetrate the crystals less effectively than Ca45, and this expectation was confirmed by the results of Table VII, in which the ratio of removable Ca45 to removable Sr8j was reduced almost one-half by heating near 500°C. Increased thermal agitation should decrease the discrimination of the crystal lattice between Ca and Sr ; Collin (16), in fact, found that heating at 950°C. produced a uniform distribution of Ca and Sr in crystals obtained from inhomogeneous strontium-calcium hydroxyapatite precipitates. A decrease to room temperature, on the other hand, should produce at least as much discrimination between the two elements as was found at 500°C.
174
SAMACHSON
AND
A change in the ratio of Ca45 to SP5 should therefore be a better criterion of penetration into the crystals than merely a decrease in the amount removed. As this ratio did not decrease significantly in 8 days at room temperature, there was little or no recrystallization during this period. This is consistent with the fact that the amounts of radioisotope removable from ashed bone (Table IV) did not change with time, although it must be pointed out that after ashing, crystals of bone mineral were probably larger, possessed a smaller surface/ volume ratio, and did not acquire a genuine hydration layer on shaking with radioisotope solution. Much that others have reported as recrystallization at room temperature may have been due to changes in the organic matrix, the diffusion layer, or the bone surface. In some of the expriments of Dallemagne (15), however, where the bone was allowed to stand for periods up to 42 days, genuine recrystallization may also have been encountered. REFERENCES 1. B~~ER, G. C. H., C.IRLSSON, A., AND LINDQUIST, B., Kungl. Fisiografiska Sdllskapets Z Lund Fdrhandlingar 26, 1 (1955). 2. BLUER, G. C. H., CARLSSON, A., AND LINDQVIPT. B.. Acta Physiol. &and. 36,67 (1955).
LEDERER 3. ARNOLD, J. S., .~ND JEE, W. S. S., Proc.
Sot. Exptl. Biol. Med. 86, 658 (1954). 4. SPENCER, H., BND GM~~CHSON, J., Clinical Sci. 20, 333 (1961). 5. BACON, J. A., PATRICK, H., AND H~NS~RD, S. L., Proc. Sot. Exptl. Biol. Med. 93, 319 (1956). 6. D.~LLEMAGNE, M. J., BODSON, P., BND F~BRY, C., Biochem. Biophys. Acta 18, 394 (1955). 7. DALLEMAGNE, M.J., DEWITTE, R., .~NDF.~BRY, C., Bull. Sot. Chim. Biol. 38, 685 (195G). 8. NEUMAN, W. F., AND NEUMAN, M. W., “The Chemical Dynamics of Bone Mineral,” p. 63. Univ. Chicago Press, Chicago, Illinois, 1958. 9. DALLEMAGNE, M.J., BODSON, R., .IND UETEN, J., Bull. Sot. Chim. Biol. 40, 233 (1958). 10. RICHELLE, L., AND DALLEM~~GNE, IQ. J., Bull. Sot. Chim. Biol. 4.0, 1133 (1958). 11. BLAU, M., SPENCER, H., SWERNOV, J., AND LASZLO, D., Science 120, 1029 (1954). 12. FISKE, C. H., SND SUBB.IROW, T., J. Biol. Chem. 66, 375 (1925). 13. S~MUHSON, J., .&ND LEDERER, H., Arch. Biochem. Biophys. 88, 355 (1960). 14. SNEDECOR, G. W., “Statistical Methods,” 5th ed., p. 38. Iowa State College Press, Ames, Iowa, 1956. 15. DALLEMAGNE, M. J., SND RICHELLE, L., Reo. Ferment. Industries Alimentaries 13, 147 (1958). 16. COLLIN, R. L., J. Am. Chem. Sot. 81, 6275 (1959).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
The Removal JOSEPH From the Veterans
lo.?,
168-174 (1963)
of Srs and Ca45 from SAMACHSON
Administration
Hospital,
AND
Hines,
Received
March
Bone in Vitro’
HILDA Illinois;
LEDERER
and Montefiore
Hospital,
New York
21, 1963
Defatted bone powder, anorganic bone, ashed bone, and apatite which had taken up Ca45 and Sr*6 under different conditions were subjected to wash solutions after standing for different lengths of time or after being subjected to heat treatment. A number of factors affected the ratio of Cad5 removed to SF removed, but under a given set of conditions a change in the ratio could be used as a criterion of recrystallization. The decrease in amounts removable after different periods of standing could be ascribed primarily to processes occurring in the organic matter of bone, and to a lesser extent in the hydration layer and on the crystal surface. There was no evidence of genuine recrystallization at room temperature.
The retention of radioisotopes is a problem which has become of increasing concern in recent years and has aroused the interest of many investigators. Radioactive isotopes of strontium and calcium are taken up by the skeleton, at first mostly by exchange with the calcium of already formed bone, and later on chiefly by the accretion of new bone (1, 2)) becoming increasingly difficult to remove as the length of time after administration increases (3,4). After accretion, such potentially dangerous isotopes as SrgO are firmly fixed in the hard tissues and can be removed only by resorption during the normal processes of bone remodeling, or by the dissolution of bone with such agents as ammonium chloride (4) or parathyroid hormone (5). Although Srs5 and Ca45 are relatively easy to remove before accretion has taken place, even exchange may not be completely reversible. Exchange with the calcium of the bone surface must be preceded by the diffusion of calcium or strontium through the organic matrix of bone (6,7) and through a hydration layer of the hydroxyapatite crystals (8), and it may be followed by 1 This National Diseases,
incorporation of the radioisotopes into the interior of the crystals. The physical chemical processes involved are complicated, occurring to a large extent simultaneously with each other and with the physiological processes of accretion and resorption, and they are intimately connected with the still uncertain mechanisms of bone formation. The removal of radioisotopes from bone requires a reversal of these processes. Dallemagne and his co-workers (9, 10) have used Ca45 to study recrystallization and throw light on the nature of bone and bone mineral. In the present study, Sr85 has also been utilized, not only to supply additional data, but because the Ca45/Sr85 ratio of uptake or release under different conditions provides information that the values for individual radioisotopes do not. The effects of time and temperature on bone which had taken up Ca45 and SF in vitro under different conditions prior to the removal of the radioisotopes have been investigated in the hope of distinguishing between diffusion through the organic matrix, diffusion through the hydration layer, and recrystallization. EXPERIMENTAL
work was supported by grant A-5572, Institute of Arthritis and Metabolic National Institutes of Health.
Bone was prepared from canine tibiae which were dried, ground to between 60 and 100 mesh, 168
REMOVAL
OF SI?J AND
Ca46 FROM
TABLE
BONE
IN VITRO
169
I
EFFECT OFTIME ON REMOVAL OF Ca4” AND Sr*6~~~~ BONE POWDERBY BUFFER SOLUTION Carrier in original radioisotope solution None None Ca Ca Sr Sr + Ca Sr + Ca Ca + P Ca + P Sr + P Averages
% of bone radioisotope removed by buffer Immediately SP Ca45/Sr”6 Ca4” 25.4 28.5 32.6 25.5 31.3 35.4 36.9 38.0 38.0 38.2 32.98
34.8 38.4 42.0 32.7 46.0 45.3 48.2 44.8 45.5 44.2 42.19
0.73 0.74 0.78 0.78 0.68 0.78 0.77 0.85 0.84 0.86 0.781
After 72 hr. SP Ca43/Sr85 C&s
Cd6 13.2 13.6 14.7 12.5 14.0 14.3 10.2 11.4 22.4 14.03
defatted, and dried again at 105°C. To obtain “anorganic” bone, part of the crushed bone was refluxed with ethylenediamine, washed with methyl alcohol, and dried. Ashed bone was obtained by heating crushed bone for about 2 hr. at 550°C. For purposes of comparison with ashed bone, part of a single apatite crystal2 was crushed and ground to below 200 mesh. SrssC12 without carrier and Ca4%12 (specific activity of approximately 20-30 PC. per ml.) were used. Solutions buffered with 0.04 X sodium Verona1 and hydrochloric acid to a pH of approximately 7.3 were prepared containing Sr*S and Ca45, either without added carrier, with CaC12, or SrCls as carrier, or with both. In shaking with bone powder and ethylenediamine-treated bone, carriers were used at a concentration of 2.5 mM; in shaking with apatite and ashed tibia, at 1.25 mM. In some cases, sodium phosphates were added to give a final concentration of 3 mg% P. One hundred-mg. samples of ground bone, ashed bone, or apatite were “anorganic bone,” treated at 37°C. for 2 hr. with 50 ml. of solution in flasks which were’ attached to a mechanical stirrer and rotated to give the effect of gentle shaking. The sample was then removed and washed by shaking with fresh W-ml. portions of solution, usually containing buffer alone at a pH of 7.3. The effect of washing with water was compared with that of washing with 2.5 mM CaClg solution in a 2 x 2 x 2 factorial experiment, in which the other factors were bone powder vs. “anorganic” 2 This apatite had a high fluorine content and was obtained through the courtesy of Clifford Frondel, Professor of Mineralogy, Harvard University.
20.0 20.7 22.3 17.4 15.2 18.6 15.4 13.3 26.4 18.81
0.66 0.66 0.66 0.72 0.92 0.77 0.66 0.86 0.85 0.751
5.9 8.1 7.9 10.3 10.9 12.6 11.0 16.4 10.39
After 192hr.
se
Ca46/S19"
10.4 15.2 20.0
0.57 0.53 0.40
13.8 13.8 14.9 12.3 20.9 15.16
0.75 0.79 0.85 0.89 0.78 0.695
bone powder, and uptake of radioisotopes in the presence of 2.5 mM CaCl* carrier or its absence. In some experiments, the bone samples, after taking up radioisotopes, were heated at approximately 475” or 525°C. for varying lengths of time, while in others they were allowed to stand without drying for 0, 72, or 196 hr. They were then shaken with the buffered wash solutions indicated above, and these were analyzed for Ca4S and Srss. The original solutions were also analyzed, after shaking with bone samples, for P, and if no stable Sr was present, for stable Ca. Bone samples were ashed, unless organic matter had been removed from them, dissolved in HCI, and also analyzed for Ca and P, as well as for Ca4h and SrsS. Experiments were usually performed in duplicate. Srs5 was determined in a scintillation counter of the well type, Ca45 by precipitation with stable Ca, as previously reported (II), counting being done in a thin-window gas flow counter. Phosphorus was determined by the method of Fiske and SubbaRow (12), stable calcium by precipitation with ammonium oxalate, and titration of the dissolved precipitate with potassium permanganate. RESULTS
Table I shows the effect of time on the removal of Ca45 and SP taken up by bone from various solutions. From 25 to 38% of the Ca45 and 33 to 48% of the Srs5 could be washed out by buffer solution used immediately. After 72 hr., the average percentage of removable radioisotope had decreased by more than half. After 120
170
SAMACHSON
AND TABLE
EFFECT OF TIME
II
ON REMOVAL OF Cad5 AND Sr85 FROM ANORGANIC BONE BY BUFFER SOLUTIONS ‘% bone radioisotope removed by buffer
Carrier in original radioisotope solution
After 72 hr.
Immediately C@
srss
None Ca Ca Ca + Sr Ca + Sr Sr Sr
16.2 18.4 18.7 19.6 17.9 17.6 16.9 16.7
22.2 25.1 26.6 27.3 26.6 26.3 28.6 27.5
0.73 0.73 0.70 0.72 0.67 0.67 0.59 0.61
13.0 12.3 13.3 12.9 14.3 12.5 14.1 11.5
Ca + P Ca + P Sr + P Sr + P Averages
15.9 14.9 20.2 18.4 17.62
21.7 21.5 29.1 26.9 25.78
0.73 0.69 0.69 0.68 0.684
9.9 9.9 12.3 11.0 12.25
None
CaJ5jSrs~
CC&
TABLE III UPTAKE OF RADIOISOTOPES BY BONE POWDER AND BY ANORGANIC BONE ‘% of radioisoto~;ae&riginal
solution
Carrier C&4’
Bone powder None (6)= Ca (4) Sr (2) Sr + Ca (6) Ca + P (6) Sr + P (3) Anorganic bone None (6) Ca (6) Ca + Sr (4) Sr (4) Ca + P (6) Sr + P (6) a Number
LEDERER
Sr86
CaWSlg~
23.5-25.618.4-21.1 7.6-15.0 6.0-12.1 19.8-20.0 8.7, 9.2 8.2-10.0 5.3- 6.2 15.8-17.912.0-13.0 20.4-21.4 9.1- 9.5
1.20-1.32 1.24-1.40 2.17,2.28 1.5c1.72 1.31-1.43 2.15-2.35
49.5-64.241.4-55.9 29.5-35.020.9-27.2 23.7-26.212.2-13.9 40.3-43.818.0-21.7 33.G48.425.3-38.2 47.&50.621.6-24.5
1.15-1.20 1.29-1.36 1.88-1.94 2.02-2.24 1.26-1.35 1.99-2.21
of replicates
in parentheses.
additional hr., there was a further highly significant decrease of almost 4% in removable Ca45 and SF. The ratio of Ca45/ Srs5 did not change significantly with time. More Ca45 could be removed from the samples which had been shaken with phosphate than from those not shaken with phosphate, the difference being significant at the 5 % level. There was no significant
After 192 hrs.
.caqws
C@
SC
Ca”5jSF
18.9 17.3 18.2 17.3 24.6 20.1 23.3 20.1
0.69 0.71 0.73 0.75 0.58 0.62 0.61 0.57
12.0 13.1 11.5 12.8 -
16.1 16.7 15.9 17.7 -
0.75 0.78 0.72 0.72 -
14.4 14.7 21.7 19.9 19.21
0.69 0.67 0.57 0.55 0.645
10.1 10.0 13.6 14.6 12.21
14.1 13.8 21.3 22.1 17.21
0.72 0.72 0.64 0.66 0.714
Sldb
difference in Srsj washed out, but the ratio of Ca45/SF was significantly higher at the 1% level after the use of phosphate, both for samples washed immediately and for all samples washed. Results for anorganic bone are presented in Table II. Smaller fractions of the Ca4j and Sr*5 taken up were removed by immediate washing. However, as anorganic bone took up more than twice as much radioisotope as untreated bone (Table III), the amounts removed, expressed as fractions of the radioisotope originally available for uptake by equivalent amounts of bone salt, were about equal. At 72 hr., the average amounts removed had decreased by about a third. An analysis of variance showed that the effects of both time and treatment were highly significant, the significance of treatment being due chiefly to the calcium carrier plus phosphate. In contrast to what is seen in Table I, the amounts washed out after this treatment were significantly lower. There was no further decrease of removable radioisotope at 192 hr. The ratio of removable Ca4j/SrS5 depended on pretreatment. It appeared to decrease during the first 72 hr. and then rise again. The differences were small and probably not significant, although the unusually small variability of duplicate samples made them appear significant.
REMOVAL
OF SP
AND
Ca46 FROM
TABLE EFFECT
OF TIME
ON REMOVAL
BONE
IN
171
VZ’Z’RO
IV
OF Ca46 AND Sra5 FROM AMIED
TIBIA
BY BUFFER
SOLUTION
Y0 bone radioisotope removed by buffer Carrier in original radioisotope solution
None (2)a Ca (2) Sr (2, I)* Ca + Sr (4) Averages (10, 12)c
29.7 35.7 45.2 34.2 35.73
a Number of replicates * Immediate treatment c Averages of individual
After 72 hrs.
Immediately
in parentheses. in duplicate; after values.
39.2 44.2 52.4 35.5 41.33
0.76 0.81 0.87 0.97 0.874
35.5 29.1 32.1 29.7 31.33
42.3 34.8 40.9 32.3 37.23
0.84 0.84 0.79 0.95 0.855
72 hr. in quadruplicate.
possibly because error was greater when small amounts were involved. The data for apatite are similar and are therefore not listed in detail. There was even W~/S+ taken Cd6/Srss in Carrier in original up (% original wash solution less uptake and more variability, and again solution (% in bone) solution) no change with time. The average Ca45/Sr85 ratio in the wash solutions was greater than None (4)5 1.30 0.89 1. Table V shows, however, that these Ca (4) 1.13 0.99 Sr (4) 1.51 1.25 ratios were less than the corresponding Ca + Sr (12) 1.12 1.03 Ca45/Sr85 ratios for bone uptake, i.e., the apatite continued to show some preferential a Xumber of replicates in parentheses. retention of Ca45. The data of Table VI indicate that wash stable Ca removed The uptake of radioisotope by bone from solutions containing the original solution equalled the sum of about twice as much Ca45 and SrE5 as solubuffer alone. Again a radioisotopes found in bone and in the wash tions containing was solution, and the values listed for Ca45, larger fraction of the radioisotope SF, and their ratios in Table III agree removed from untreated than from anorganic bone. The ratio of Ca4j/Srs5 was very with those found when no wash solution was used and the bone was analyzed for significantly greater when calcium was used in the wash water and was also greater for total uptake (13). As the number of replicates varied from 2 to 6, ranges are given untreated than for anorganic bone. Amounts instead of standard deviation, although the washed out were the same whether the latter can easily be estimated from the uptake of radioisotope had been from solutions containing buffer alone or buffer plus former (14). Ashed bone took up smaller amounts of calcium. The fraction of Ca45 and SP removable radioisotope from the original solution, calcium averaging 25 % Ca45 and 20 % SP in the with wash solutions containing absence of carrier, and 7.2 % Ca45 and 4.3 % decreased when the bone had been heated (Table VII). On one sample of bone heated Sr85 in the presence of calcium plus stronat 475°C. for 2 hr. the effect was minor, tium. About a third of the Ca45and slightly possibly because of failure to attain the more of the SP could be washed out (Table temperature soon enough. The other data IV). There was no significant change with time in Ca4j, Sr85, or Ca45/Sr85 ratios, the indicate no difference in amounts removable latter being higher than in the case of whether time of heating was 2, 6, or 24 hr. An increase in temperature to 525°C. untreated or anorganic bone. Variability was greater than in the previous cases, reduced the amount removable by half. TABLE
RATIOS
V
OF Ca4s/Sr85 TAKEN UP BY APATITE WASHED OUT BY BUFFER
AND
172
SAMACHSON
AND TABLE
EFFECT
OF CONDITIONS
of radioisotopes
Washed without
Ca
Ca
Anorganic bone Washed without Washed with
Ca
Ca
Removal Powdered bone Washed without Washed with
with
TABLE
475°C.
24 hr.
525°C.
2 hr. 6 hr. 24 hr.
Ca
Shaken with
Ca
Ca45 14.5 12.2 12.2 12.1
Srs5 10.1 9.5 9.2 9.3
Ca46/SrsS 1.44 1.28 1.33 1.30
63.5 61.6 56.5 54.0
51.5 49.6 48.9 49.1
1.23 1.24 1.16 1.10
39.2 39.2 37.9 36.7
27.7 28.3 27.1 26.4
1.42 1.39 1.40 1.39
from bone,
70 in bone
24.3 23.1 49.3 45.5
30.8 29.0 55.2 50.2
0.79 0.80 0.89 0.91
26.9 27.9 51.6 47.1
33.7 31.6 51.1 48.4
0.80 0.88 1.01 0.97
Ca
14.8 15.7 39.1 35.0
21.0 22.0 45.0 41.1
0.70 0.71 0.87 0.85
14.8 15.3 34.8 36.0
21.3 21.9 42.1 44.7
0.69 0.70 0.83 0.81
Ca
VII
23.08 20.83 20.14
6 hr.
solution
Ca45/SrsS 1.27 1.31 1.28 1.24
y. removed
2 hr.
ON
Ca
Cd5
None
SOLUTION
Srs5 26.3 25.5 23.9 25.1
EFFECT OF HEATING OF BONE ON SUBSEQUENT REMOVAL OF SrsS AND Ca45 BY WASHING Heating
70 in original
IN WASH
Ca4& 33.3 33.3 30.6 31.2
of radioisotopes
Ca
Anorganic bone Washed without Washed
by bone,
Shaken without
bone
Washed with
VI
OF UPTAKE AND PRESENCE OF CARRIER REMOVAL OF Ca45 AND Srs5 FROM BONE
Uptake Powdered
LEDERER
5.89 17.34 6.07 6.08 5.63 5.81
2.89 2.74 2.98
sr=
Ca46/Sr85
27.36 22.09 21 .OY
0.84 0.91 0.95
Average
0.90
10.40 24.05 12.17 12.32 11.82 11.78
0.57 0.72 0.50 0.49 0.48 0.49
Average
0.54
5.78 4.21 5.12
0.50 0.65 0.58
Average
0.58
Again with the indicated exception, the ratio of Ca45/Sr85 removed was about 0.5, the same at both temperatures and various times of heating, and very significantly different from the value of 0.9 before heating. DISCUSSION
-
A comparison of the considerable effect of standing for 72 hr. on powdered whole bone (Table I) with the smaller effect on bone which lacks organic matter (Table II) indicates that processes occurring in the organic matrix are of major importance in determining the extent to which radioisotopes can be removed. During these 72 hr. the radioisotopes either diffuse through the organic matrix to the crystals (6, 7) or are more firmly bound by the organic matrix itself. The former hypothesis appears more likely, but the latter cannot be completely excluded. Previous data obtained on the uptake of radioisotopes by organic matter left when bone is decalcified with ethylenediaminetetraacetic acid (13) have
REMOVAL
OF Sr*s AND
C a45 FROM
been used to calculate the amounts of Ca45 and Sr86 taken up by organic matter in the present study. Although these amounts are too small to account for the changes that take place in 72 hr. in Table I, the original untreated organic matter may have a greater capacity to bind these elements, and the organic-inorganic interface may also be involved. Dallemagne has repeatedly emphasized (6, 7) that the mineral in the organic bone differs from the isolated bone mineral, the organic matter apparently stabilizing the mineral structure. Conversely, the mineral may stabilize the organic structure and permit greater uptake of radioisotopes. A comparison of the effect of standing on ethylenediamine-treated bone (Table II) with the lack of effect on ashed bone (Table IV) indicates that the hydration layer also takes part in controlling the removal of radioisotopes, although to a lesser extent than the organic matrix. This layer serves as a medium for the dissolution and redeposition of calcium and phosphate ions from the crystal surface and for the exchange of ions which enter from the bulk solution (8). Kot only calcium and strontium but sodium, magnesium, and hydronium ions of bone may exchange with calcium or strontium from the solution and defects in the crystal surface may be created or eliminated. Although these processes affect the amount of radioisotope removable, and may be considered a form of recrystallization, they differ considerably from the recrystallization which involves penetration into the crystal interior. They differ also from the recrystallization which Arnold and Jee (3) reported to take place in viva. The latter involves the physiological dissolution of bone and the formation of new bone. The ratio of removable Ca45 to Srs5 depended on the nature of both the solutions from which the radioisotopes were taken up and the bone powder. The changes in amounts washed out after uptake from solutions containing Ca and P is of special interest. In this case, the amounts removable from powdered bone as well as the ratio of removable Ca45 to Srs5 were greater than after uptake from buffered solutions. On
BONE
IN VITRO
173
the other hand, the amounts removable from anorganic bone were lower after uptake from Ca and P solutions than after uptake from buffered solutions. The use of Ca and P in the amounts indicated probably led to the deposition of calcium phosphate, In whole bone this may have been in the form of colloidal tiny particles which deposited in the organic matrix and were easily dissolved again, while in anorganic bone, new layers may have been added to already existing apatite crystals and may have been more difficult to remove. The recrystallization of bone and bone mineral has been studied by a number of investigators, including Arnold and Jee (3), who washed bone with distilled water and calcium acetate solutions, and Dallemagne and his coworkers (6, 7, 9), who carried out numerous experiments with hydrochloric acid. Dallemagne used as his criterion of recrystallization the pattern of removal with successive washes of acid, assuming that these would more or less regularly attack successive layers of bone salt crystal. Both investigators found that removal of the radioisotope became more difficult as time after deposition increased, and concluded that recrystallization was taking place. Difficulty of removal alone, however, is not a sufficient criterion of recrystallization, and another one was sought for this study, based on Dallemagne’s observation that heating at 400°C. for 24 hr. was sufficient for Ca45to penetrate uniformly into the interior of bone salt crystals (15). It was expected that Sr85, because of its larger radius, would penetrate the crystals less effectively than Ca45, and this expectation was confirmed by the results of Table VII, in which the ratio of removable Ca45 to removable Sr8j was reduced almost one-half by heating near 500°C. Increased thermal agitation should decrease the discrimination of the crystal lattice between Ca and Sr ; Collin (16), in fact, found that heating at 950°C. produced a uniform distribution of Ca and Sr in crystals obtained from inhomogeneous strontium-calcium hydroxyapatite precipitates. A decrease to room temperature, on the other hand, should produce at least as much discrimination between the two elements as was found at 500°C.
174
SAMACHSON
AND
A change in the ratio of Ca45 to SP5 should therefore be a better criterion of penetration into the crystals than merely a decrease in the amount removed. As this ratio did not decrease significantly in 8 days at room temperature, there was little or no recrystallization during this period. This is consistent with the fact that the amounts of radioisotope removable from ashed bone (Table IV) did not change with time, although it must be pointed out that after ashing, crystals of bone mineral were probably larger, possessed a smaller surface/ volume ratio, and did not acquire a genuine hydration layer on shaking with radioisotope solution. Much that others have reported as recrystallization at room temperature may have been due to changes in the organic matrix, the diffusion layer, or the bone surface. In some of the expriments of Dallemagne (15), however, where the bone was allowed to stand for periods up to 42 days, genuine recrystallization may also have been encountered. REFERENCES 1. B~~ER, G. C. H., C.IRLSSON, A., AND LINDQUIST, B., Kungl. Fisiografiska Sdllskapets Z Lund Fdrhandlingar 26, 1 (1955). 2. BLUER, G. C. H., CARLSSON, A., AND LINDQVIPT. B.. Acta Physiol. &and. 36,67 (1955).
LEDERER 3. ARNOLD, J. S., .~ND JEE, W. S. S., Proc.
Sot. Exptl. Biol. Med. 86, 658 (1954). 4. SPENCER, H., BND GM~~CHSON, J., Clinical Sci. 20, 333 (1961). 5. BACON, J. A., PATRICK, H., AND H~NS~RD, S. L., Proc. Sot. Exptl. Biol. Med. 93, 319 (1956). 6. D.~LLEMAGNE, M. J., BODSON, P., BND F~BRY, C., Biochem. Biophys. Acta 18, 394 (1955). 7. DALLEMAGNE, M.J., DEWITTE, R., .~NDF.~BRY, C., Bull. Sot. Chim. Biol. 38, 685 (195G). 8. NEUMAN, W. F., AND NEUMAN, M. W., “The Chemical Dynamics of Bone Mineral,” p. 63. Univ. Chicago Press, Chicago, Illinois, 1958. 9. DALLEMAGNE, M.J., BODSON, R., .IND UETEN, J., Bull. Sot. Chim. Biol. 40, 233 (1958). 10. RICHELLE, L., AND DALLEM~~GNE, IQ. J., Bull. Sot. Chim. Biol. 4.0, 1133 (1958). 11. BLAU, M., SPENCER, H., SWERNOV, J., AND LASZLO, D., Science 120, 1029 (1954). 12. FISKE, C. H., SND SUBB.IROW, T., J. Biol. Chem. 66, 375 (1925). 13. S~MUHSON, J., .&ND LEDERER, H., Arch. Biochem. Biophys. 88, 355 (1960). 14. SNEDECOR, G. W., “Statistical Methods,” 5th ed., p. 38. Iowa State College Press, Ames, Iowa, 1956. 15. DALLEMAGNE, M. J., SND RICHELLE, L., Reo. Ferment. Industries Alimentaries 13, 147 (1958). 16. COLLIN, R. L., J. Am. Chem. Sot. 81, 6275 (1959).