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Effect of fluoride on bone “crystallinity” in the growing rat
SHORT COMMUNICATIONS
565
sc 2152
Effect of fluoride on bone "crystallinity" in the growin 9 rat The " c r y s t a l l i n i t y " of human bone as defined by the degree of resolution of the four principal reflections of the X-ray-diffraction patterns improves with the increase in fluoride content z. I m p r o v e m e n t in crystallinity implies crystal growth and/or an annealing out of the structural imperfections of the crystals. Previous data on experimental animals indicate equivocation over the effect of fluoride on the crystallinity of bone apatite2, 3. Since the relation between duration of fluoride exposure and crystallinity was not delineated in the human study, the present investigation was made to study the effect of different levels of fluoride over various time periods on crystal texture in rat bones. At 47 days of age, four groups of male rats received o, io, 50 and IOO parts per million F-, respectively, in the drinking water. Animals were maintained on the diet of ESTREMERA et al. 4, containing less than 0.8 part/million F - and modified by the incorporation of a vitamin mixture into the feed. Animals were sacrificed at 47, 72, lO2 and 161 days of age. X-ray-diffraction patterns (copper K-~ radiation) were taken of the dry fat-free powders of the tibia-fibula, mandible and vertebra which had been passed through a 2oo-mesh sieve. Care was taken to avoid orientation effects. A Geiger-counter diffractometer with a high-resolution slit system was used. The degree of resolution of the 4 principal reflections of the X-ray patterns (Miller indices: 211,112, 3o0, 202) was used as a measure of the "crystallinity" of each specimen and was expressed as/5, the estimated average width at half m a x i m u m of the reflections. The/5 values were estimated for each X-ray pattern b y matching each with one of a series of IO templates of different/5 values. Each template was the sum of the four reflections approximated in shape as gaussian probability functions with equal/5 values. In all templates, each reflection was centered at the 2~ value and was given the relative intensity value of the well-resolved hydroxyapatite X-ray pattern. For chemical analysis fluoride was distilled from samples b y the method of SINGER et al. 5 and determined b y the colorimetric procedure of MEGREGIAN6. It m a y be seen from Table I that the fl value of rat-bone apatite is a function of both the duration of exposure to the F - supplement and its concentration. There was no significant change in "crystallinity" with age in the 3 bones from rats receiving no F-. In addition, the administration of IO, 50 and ioo parts/million F - affected tile /3 values of the various bones to different degrees. Thus, the vertebra did not show a n y pronounced increase in crystallinity (decrease in/5 value) until the animals had received IOO parts/million F - for 114 days. The tibia-fibula showed essentially no change in line broadening in animals receiving IO, 50 or IOO parts/million F - for 25 days. The mandibles showed first evidence of an increase in crystallinity in rats receiving IOO parts/million F - for 25 days. Thereafter, the tibia-fibulae and the mandibles showed similar changes in /5 values, in that the increase in crystallinity was a function of not only the concentration of F in the bones but also the duration of exposure. In the rat there appears to be a time lag before the influence of the F - is sufficient to affect crystallinity of the bone apatite. For example, tibia-fibulae containing o.4ol % F at 72 days show no essential change in /5 value from the 47-day group whereas tibia-fibulae containing only o.133 % F show a change at lO2 days. At Biochim. Biophys. Acta, 64 (1962) 565-567
566
SHORT COMMUNICATIONS TABLE
I
RELATION BETWEEN CRYSTALLINITY (•), L E N G T H OF EXPOSURE TO FLUORIDATED WATER AND FLUORINE CONTENT OF THE VERTEBRA, MANDIBLE AND TIBIA-FIBULA OF THE GROWING RAT
Cohen. (parts/million) 17- in drinking water Age (days)
o No.
Vertebra 47
F - (%)*
3
0.070
zo ~**
No.
F - (%)*
5° fl**
No.
F - (%J*
:*oo fl**
No.
F - (%)*
~**
1.2o
~20.OIO
72
3
lO2
0.066 :t- 0 . o 0 3 I 0.o60
1.2o
161
3
0.097 o.o15
1.2o
Mandible 47
3
o.o7 o 0,005 o.o53 O.Ol 4 o.048
1.2o
i 72
3
lO2
I
161
3
1.2o
L2o 1.2o
0.097 o.034
i.io
1.16
lO2
o.o41 ± 0.006 3 o.o5o o.ooi i 0.062
1.2o
161
3
1.16
Tibia-fibula 47 72
3
o.o61 ± o.o12
1-14
4
o.13o ~_ o . o 1 3 3 °.151 0.022 3 o.167 ~ 0.o05
4
o.lo2 ~ 0.003 3 o.126 :£ 0 . o 0 7 3 o-159 J_ O.Ol 7
4
o.117 -E 0 . 0 0 9 3 o.133 o.o14 3 o.135 ~_ 0 . 0 0 9
1.2o I.~7 1.13
1.2o
4
O.277 ± O.O27 2 o.323 ± 0.112 3 O.481 ± O.O49
4
o.296 ~- o . o 1 6 2 o.388 ~- o . 0 3 2 3 o.555 ~2 O.lO 3
1.2o 1.13
1.12
4
o.285 0.022 i 0.373 -L o . 0 6 6 3 0.385 ± 0.086
1.16 1.13 I.IO
1.o9 1.o 5 0.98
i.io
i
0.98 0.98
* All fluoride values expressed on ash basis as mean ** A v e r a g e d e v i a t i o n f r o m m e a n ± 5 % .
~ standard
1.o4 0.92
3
°,466 :£ 0.O77 2 o.661 :~ O.Ol 3 3 °.567 ~ 0.O43
3
o.472 ~ 0.072 2 o.555 ~ 0.055 3 0.608 ~ O.Oli
2
o.4oi ~ 0.078 2 o.51o ~ o.o76 3 o.733 ~ 0.080
I.t 7 1.10
1.0 3
1 .OO
1.03
0.8 5
I ,I0
0.92 o,88
error of mean.
161 days all 3 bones show an increase in crystailinity as their F concentration increased. I t is interesting to observe t h a t the vertebra showed the least change in crystallinity although the concentration of F in all the bones examined was essentially similar at comparable time periods. The reason for this is not apparent at present since it has been shown t h a t spongy bone contains smaller crystals than compact bone, dentin or enamel, and that the degree of resolution of the X-ray-diffraction pattern and hence the crystallinity increases in the same order 7. More recently, MENCZEL et al. s have reported t h a t in adult rats the shaft of the tibia is more crystalline than the ends, and that both shafts and ends improve in crystallinity with increasing exposure to F-. In the present experiment, the rats on no F - show little or no difference in crystallinity in the bones studied even though the relative proportion of spongy to compact bone probably differs in vertebra, tibia-fibula and mandible. Biochim. Biophys. Acta, 6 4 (1962) 5 6 5 - 5 6 7
567
SHORT COMMUNICATIONS
It appears from the X-ray-diffraction results that fluoride in some way reduced the effective specific surfaces of the bone mineral by increasing the crystal size and/or decreasing the crystal imperfections. Therefore, ions such as citrate which occupy only crystal surface positions in apatites 9 would be decreased in concentration in fluoridated bone due to the reduction in effective specific surface. This phenomenon may explain the inverse relation between fluoride and citrate in both rat TM and human bone n. This study was supported in part by a research grant (D-IooI) from the National Institutes of Health of the Public Health Service to H.S. and R.S.
* Biophysics Laboratory, Department of Physics, The Pennsylvania State University, University Park, Pa., and ** National Institute of Dental Research, National Institutes of Health, Bethesda, Md. (U.S.A.)
HAROLD SCHRAER* A . S. POSNER** ROSEMARY SCHRAER* I. ZIPKIN* *
1 I. Z1PKIN, A. S. POSNER AND E. C. EANES, Biochim. Biophys. Acta, 59 (1962) 255. a M. REYNOLDS, K. E. CORRIGAN, H. S. HAYDEN, I. G. MACY AND H. A. HUNSCHER, Am. J . Roentgenol. Radium Therapy, 39 (1938) lO3. 8 S. J. FUZlE, J. Mgr. Chem. Soc. Japan, 35 (1959) 155. 4 H. ~R. ESTREMERA AND W. D. ARMSTRONG, J. Nutrition, 35 (1948) 611. 5 L. SINGER AND W. D. ARMSTRONG, Anal. Chem., 26 (1954) 9o4. S. MEGREGIAN, Anal. Chem., 26 (1954) 1161. R. C. LIKINS, A. S. POSNER, ]3. PARETZKIN AND A. P. FROST, J. Biol. Chem., 236 (1961) 2804. 8 j. MENCZEL, A. S. POSNER, H. SCHRAER, G. PAKIS AND R. C. LIKINS, Proc. Soc. Exptl. Biol. :~Ied., IiO (1962) 6o9. s W. F. NEUMAN ANn M. W. NEUMAN, The Chemical Dynamics of Bone Mineral, University of Chicago Press, 1958 . 10 I. ZIPKIN, W. A. LEE, R. SCHRAER AND H. SCHRAER, Vth Intern. Congr. Biochem., Moscow, i96I, Abstracts of Communications, Pergamon Press, New York, 1961. 11 I. ZIPKIN, F. J. McCLuRE AND W. A. LEE, Arch. Oral Biol., 2 (196o) 19o.
Received June 7th, 1962 Biochim. Biophys. Acta, 64 (1962) 565-567
565
sc 2152
Effect of fluoride on bone "crystallinity" in the growin 9 rat The " c r y s t a l l i n i t y " of human bone as defined by the degree of resolution of the four principal reflections of the X-ray-diffraction patterns improves with the increase in fluoride content z. I m p r o v e m e n t in crystallinity implies crystal growth and/or an annealing out of the structural imperfections of the crystals. Previous data on experimental animals indicate equivocation over the effect of fluoride on the crystallinity of bone apatite2, 3. Since the relation between duration of fluoride exposure and crystallinity was not delineated in the human study, the present investigation was made to study the effect of different levels of fluoride over various time periods on crystal texture in rat bones. At 47 days of age, four groups of male rats received o, io, 50 and IOO parts per million F-, respectively, in the drinking water. Animals were maintained on the diet of ESTREMERA et al. 4, containing less than 0.8 part/million F - and modified by the incorporation of a vitamin mixture into the feed. Animals were sacrificed at 47, 72, lO2 and 161 days of age. X-ray-diffraction patterns (copper K-~ radiation) were taken of the dry fat-free powders of the tibia-fibula, mandible and vertebra which had been passed through a 2oo-mesh sieve. Care was taken to avoid orientation effects. A Geiger-counter diffractometer with a high-resolution slit system was used. The degree of resolution of the 4 principal reflections of the X-ray patterns (Miller indices: 211,112, 3o0, 202) was used as a measure of the "crystallinity" of each specimen and was expressed as/5, the estimated average width at half m a x i m u m of the reflections. The/5 values were estimated for each X-ray pattern b y matching each with one of a series of IO templates of different/5 values. Each template was the sum of the four reflections approximated in shape as gaussian probability functions with equal/5 values. In all templates, each reflection was centered at the 2~ value and was given the relative intensity value of the well-resolved hydroxyapatite X-ray pattern. For chemical analysis fluoride was distilled from samples b y the method of SINGER et al. 5 and determined b y the colorimetric procedure of MEGREGIAN6. It m a y be seen from Table I that the fl value of rat-bone apatite is a function of both the duration of exposure to the F - supplement and its concentration. There was no significant change in "crystallinity" with age in the 3 bones from rats receiving no F-. In addition, the administration of IO, 50 and ioo parts/million F - affected tile /3 values of the various bones to different degrees. Thus, the vertebra did not show a n y pronounced increase in crystallinity (decrease in/5 value) until the animals had received IOO parts/million F - for 114 days. The tibia-fibula showed essentially no change in line broadening in animals receiving IO, 50 or IOO parts/million F - for 25 days. The mandibles showed first evidence of an increase in crystallinity in rats receiving IOO parts/million F - for 25 days. Thereafter, the tibia-fibulae and the mandibles showed similar changes in /5 values, in that the increase in crystallinity was a function of not only the concentration of F in the bones but also the duration of exposure. In the rat there appears to be a time lag before the influence of the F - is sufficient to affect crystallinity of the bone apatite. For example, tibia-fibulae containing o.4ol % F at 72 days show no essential change in /5 value from the 47-day group whereas tibia-fibulae containing only o.133 % F show a change at lO2 days. At Biochim. Biophys. Acta, 64 (1962) 565-567
566
SHORT COMMUNICATIONS TABLE
I
RELATION BETWEEN CRYSTALLINITY (•), L E N G T H OF EXPOSURE TO FLUORIDATED WATER AND FLUORINE CONTENT OF THE VERTEBRA, MANDIBLE AND TIBIA-FIBULA OF THE GROWING RAT
Cohen. (parts/million) 17- in drinking water Age (days)
o No.
Vertebra 47
F - (%)*
3
0.070
zo ~**
No.
F - (%)*
5° fl**
No.
F - (%J*
:*oo fl**
No.
F - (%)*
~**
1.2o
~20.OIO
72
3
lO2
0.066 :t- 0 . o 0 3 I 0.o60
1.2o
161
3
0.097 o.o15
1.2o
Mandible 47
3
o.o7 o 0,005 o.o53 O.Ol 4 o.048
1.2o
i 72
3
lO2
I
161
3
1.2o
L2o 1.2o
0.097 o.034
i.io
1.16
lO2
o.o41 ± 0.006 3 o.o5o o.ooi i 0.062
1.2o
161
3
1.16
Tibia-fibula 47 72
3
o.o61 ± o.o12
1-14
4
o.13o ~_ o . o 1 3 3 °.151 0.022 3 o.167 ~ 0.o05
4
o.lo2 ~ 0.003 3 o.126 :£ 0 . o 0 7 3 o-159 J_ O.Ol 7
4
o.117 -E 0 . 0 0 9 3 o.133 o.o14 3 o.135 ~_ 0 . 0 0 9
1.2o I.~7 1.13
1.2o
4
O.277 ± O.O27 2 o.323 ± 0.112 3 O.481 ± O.O49
4
o.296 ~- o . o 1 6 2 o.388 ~- o . 0 3 2 3 o.555 ~2 O.lO 3
1.2o 1.13
1.12
4
o.285 0.022 i 0.373 -L o . 0 6 6 3 0.385 ± 0.086
1.16 1.13 I.IO
1.o9 1.o 5 0.98
i.io
i
0.98 0.98
* All fluoride values expressed on ash basis as mean ** A v e r a g e d e v i a t i o n f r o m m e a n ± 5 % .
~ standard
1.o4 0.92
3
°,466 :£ 0.O77 2 o.661 :~ O.Ol 3 3 °.567 ~ 0.O43
3
o.472 ~ 0.072 2 o.555 ~ 0.055 3 0.608 ~ O.Oli
2
o.4oi ~ 0.078 2 o.51o ~ o.o76 3 o.733 ~ 0.080
I.t 7 1.10
1.0 3
1 .OO
1.03
0.8 5
I ,I0
0.92 o,88
error of mean.
161 days all 3 bones show an increase in crystailinity as their F concentration increased. I t is interesting to observe t h a t the vertebra showed the least change in crystallinity although the concentration of F in all the bones examined was essentially similar at comparable time periods. The reason for this is not apparent at present since it has been shown t h a t spongy bone contains smaller crystals than compact bone, dentin or enamel, and that the degree of resolution of the X-ray-diffraction pattern and hence the crystallinity increases in the same order 7. More recently, MENCZEL et al. s have reported t h a t in adult rats the shaft of the tibia is more crystalline than the ends, and that both shafts and ends improve in crystallinity with increasing exposure to F-. In the present experiment, the rats on no F - show little or no difference in crystallinity in the bones studied even though the relative proportion of spongy to compact bone probably differs in vertebra, tibia-fibula and mandible. Biochim. Biophys. Acta, 6 4 (1962) 5 6 5 - 5 6 7
567
SHORT COMMUNICATIONS
It appears from the X-ray-diffraction results that fluoride in some way reduced the effective specific surfaces of the bone mineral by increasing the crystal size and/or decreasing the crystal imperfections. Therefore, ions such as citrate which occupy only crystal surface positions in apatites 9 would be decreased in concentration in fluoridated bone due to the reduction in effective specific surface. This phenomenon may explain the inverse relation between fluoride and citrate in both rat TM and human bone n. This study was supported in part by a research grant (D-IooI) from the National Institutes of Health of the Public Health Service to H.S. and R.S.
* Biophysics Laboratory, Department of Physics, The Pennsylvania State University, University Park, Pa., and ** National Institute of Dental Research, National Institutes of Health, Bethesda, Md. (U.S.A.)
HAROLD SCHRAER* A . S. POSNER** ROSEMARY SCHRAER* I. ZIPKIN* *
1 I. Z1PKIN, A. S. POSNER AND E. C. EANES, Biochim. Biophys. Acta, 59 (1962) 255. a M. REYNOLDS, K. E. CORRIGAN, H. S. HAYDEN, I. G. MACY AND H. A. HUNSCHER, Am. J . Roentgenol. Radium Therapy, 39 (1938) lO3. 8 S. J. FUZlE, J. Mgr. Chem. Soc. Japan, 35 (1959) 155. 4 H. ~R. ESTREMERA AND W. D. ARMSTRONG, J. Nutrition, 35 (1948) 611. 5 L. SINGER AND W. D. ARMSTRONG, Anal. Chem., 26 (1954) 9o4. S. MEGREGIAN, Anal. Chem., 26 (1954) 1161. R. C. LIKINS, A. S. POSNER, ]3. PARETZKIN AND A. P. FROST, J. Biol. Chem., 236 (1961) 2804. 8 j. MENCZEL, A. S. POSNER, H. SCHRAER, G. PAKIS AND R. C. LIKINS, Proc. Soc. Exptl. Biol. :~Ied., IiO (1962) 6o9. s W. F. NEUMAN ANn M. W. NEUMAN, The Chemical Dynamics of Bone Mineral, University of Chicago Press, 1958 . 10 I. ZIPKIN, W. A. LEE, R. SCHRAER AND H. SCHRAER, Vth Intern. Congr. Biochem., Moscow, i96I, Abstracts of Communications, Pergamon Press, New York, 1961. 11 I. ZIPKIN, F. J. McCLuRE AND W. A. LEE, Arch. Oral Biol., 2 (196o) 19o.
Received June 7th, 1962 Biochim. Biophys. Acta, 64 (1962) 565-567