Pflfigers Archiv
Pfliigers Arch. 370, 77-80 (1977)
EuropeanJoumal
of~y
9 by Springer-Verlag1977
Determination of Oxytetracycline Induced Fluorescence and Autofluorescence as an Indicator of Age and Sex Differences in Rat Femur* D A R I N K A D E K A N I C , K A R L O WEBER, and K R I S T A K O S T I A L Institute for Medical Research and Occupational Health, Mo~e Pijade 158, YU-41000 Zagreb, Yugoslavia
Summary. Simple and rapid methods for the determination and detection of autofluorescence and oxytetracycline (OTC) induced fluorescence in powdered and intact bone (without previous chemical extraction of the antibiotic) were used to estimate age and sex related differences in the rat femur. The autofluorescence always had collagen characteristics; it increased with age and was not sex dependent. The intensity of O T C induced fluorescence decreased with age, being higher in males than in females. In the oldest animals, however a slight increase in the fluorescence intensity was observed, and sex differences disappeared. The spectrofluorometric measurements of intact femora indicate a higher O T C retention on the bone surface of young animals and adult males but not in adult females. The results indicate that the determination of O T C retention in the bone using our quantitative fluorometric method and the detection of O T C deposition at bone surface using a spectrofluorometric technique can be a sensitive and inexpensive tool for experimental studies of bone changes in physiological and pathological conditions.
sensitive indicators of bone changes especially in osteoporosis. In 1958 Milch and collaborators stated in one of the earliest reports on tetracycline induced fluorescence in bone that with age changes in bone tissue can be detected grossly using this technique. Detailed investigations in this field were not performed, possibly because of the difficulties involved in quantitative estimation of tetracyclines in calcified tissues. Most authors over this period used tetracyclines only as a label for studying bone formation (Frost, 1969). The rare reports on bone autofluorescence showed that this type of bone fluorescence is also age related. These data indicate that autofluorescence and tetracycline induced fluorescence might be used for studying age related changes in the bone tissue. In the present investigation, which involves the use of simple and rapid methods for both the detection and measurement of autofluorescence and oxytetracycline (OTC) induced fluorescence in powdered and intact bone (without previous chemical extraction of the antibiotic), age and sex related differences in rat femur were studied.
K e y words: Bone - O T C induced fluorescence and autofluorescence - Intensity and spectrofluorometry Age and sex relationship.
METHODS Experimental Animals. Experiments were performed on male and female albino rats from own laboratory stock. The intensity of fluorescence was determined in powdered bone samples in groups of animals aged 2-20 months, each consisting of 5-22 rats. The spectral distribution of fluorescence of powdered as well as intact femoral bone was determined in the youngest-2 month old, and in adult-12 month old animals, divided into groups of 5-19 rats each.
-
INTRODUCTION Over the last 20 years several new methods have been introduced with the aim of developing more * This study was supported in part by the "Pliva", Pharmaceutical Works, Zagreb, Yugoslavia and US Department of Agriculture, Beltsville, Maryland Send offprints requests to K. Kostial at the above address
Oxytetracyeline Treatment. The treated rats received either one intraperitoneal injection of 100 mg OTC (Geomycin, "Pliva", Zagreb) per kilogram body weight (when the intensity of fluorescence was measured) or six intraperitoneal injections of 100 mg OTC per kilogram body weight (when the spectral distribution of fluorescence was determined). Control animals were given corresponding injections of a physiologicalsaline solution (0.9 % NaC1). The animals were killed by ether anaesthesia 72 h after the last injection.
78
Pflfigers Arch. 370 (1977)
Table 1. Autofluorescence intensity of powdered femoral bones of male and female rats of different age Fluorescence intensity (arbitrary units) Age (months)
Male Female
2 6 12 (10)y (20)x (10) (10) (10)(10)
20 (10) (19)
140_+ 3 c 140_+2 d
182• 184• 3
149_+2 d 170• 3 d 147_+2 d 168_+ 3 d
Values are means and S.E. Numbers in parentheses are numbers of animals: ymales xfemales ~,d Statistically significant differences between successional age groups at 1 and 0.1 ~ level respectively
Treatment of Bone Samples. Both femora were removed and cleaned from the connective tissues. The foramen nutricium was enlarged by means of a hand borer to a diameter of 1 - 2 mm and the bone marrow was removed by centrifngation (3000 g for 30 min). The bones were dried for 15 days in the dark at room temperature and after that both femora were ground into a fine powder with a mortar. We have shown previously that under such conditions consistent results can be obtained (Dekani6, 1971). For the determination of the spectral curves of intact (unpowdered) bone the right femur was also used after being dried for 15 days at room temperature. Measurement of Fluorescent Intensity and Its Spectral Distribution. The intensity of fluorescence of powdered bone samples was measured with a photoelectric fluorometer of own construction. The light source was a mercury lamp with appropriate filters to give maximum transmission at 366 nm. Each measurement was made at 45~ to the incident beam by comparison with a standard (filter paper coated with optical white). The precision of the method was _+2 ~. In our previous study we observed a linear relationship between the dose of intraperitoneally injected OTC (dose range 12.5- 200 mg/kg) and fluorescent intensity of powdered bone samples (Dekani6 et al., 1972). The spectral distribution of fluorescence in powdered and intact bone samples was determined with a commercial spectroftuorometer (Perkin Elmer-Hitachi MPF 2A). The same light source and angle of measurement were used as in the measurement of the intensity. The spectral distribution from quinine sulphate and 3-aminophthalimide in 0.1 N sulphuric acid served as standard (Lippert et al., 1959; Argauer and White, 1964; Himel and Mayer, 1970).
RESULTS
Intensity o f Autofluorescence and 0 TC Induced Fluorescence in Powdered Bone Autofluorescence. Table 1 shows the relative intensity o f autofluorescence in p o w d e r e d bone in relation to age and sex. The results show an almost linear relationship between auto fluorescence intensity and age. Samples f r o m y o u n g e r rats always h a d a significantly lower fluorescence intensity than those f r o m older animals while within the same age g r o u p the difference between male and female animals was n o t significant.
O T C Induced Fluorescence. Table 2 shows the relative intensity o f fluorescence o f p o w d e r e d f e m o r a
after a single i.p. injection o f OTC. The intensity was significantly higher in samples f r o m y o u n g animals than f r o m the older animals o f either sex until 12 m o n t h s of age. F r o m then onwards there was no further, change in either male or female animals. A statistically higher fluorescence intensity was generally observed in males. In the oldest animals (20 months) the intensity o f fluorescence was again slightly increased (statistically significant only in females) so that sex differences in b o n e fluorescence levels disappeared.
Spectrofluorometric Measurements of Autofluorescence and 0 TC Induced Fluorescence Powdered Bone Samples. Spectrofluorometric measurements resulted in curves o f four basic shapes: curves 1, 2, 3 and 4 as shown in Figure 1. Figure 2 shows the curves obtained for a commercial preparation (Gelatin W o r k s Ljubljana) o f gelatin-collagen (curve 1) and O T C (curve 2). P o w d e r e d bone samples f r o m untreated animals always gave curves o f the type for collagen fluorescence (Figs. I and 2, curve 1), while y o u n g O T C treated animals provided curves of the O T C type (Figs. 1 and 2, curve 2). Measurements f r o m p o w d e r e d f e m o r a o f adult O T C treated rats usually resulted in curves o f the type shown in Figure 1 curves 3 and 4 which resulted f r o m b o t h types of fluorescence (autofluorescence and O T C induced fluorescence).
Intact Femora. Intact (unpowdered) bone samples f r o m untreated animals again showed only the spectral characteristics o f collagen (Fig. 1, curve 1). The f e m o r a o f y o u n g O T C treated rats o f both sexes showed only the O T C characteristics (Fig. 1, curve 2). In adult O T C treated animals female f e m o r a showed no signs o f O T C induced fluorescence (Fig. 1, curve 1) indicating no O T C deposition at periosteal surface. M o s t male femora showed curves which resulted f r o m b o t h types o f fluorescence (curves 3 and 4 in Fig. 1) and sometimes even only the O T C spectral curve (Fig. 1, curve 2). DISCUSSION O u r results which concern the autofluorescence of the rat bone agree in general with the few data available in literature. The spectrum o f the emitted light has collagen characteristics, as in b o n e samples f r o m h u m a n s ( B a c h m a n and Ellis, 1965; A r m s t r o n g and Horsley, 1972), confirming an earlier report that the colour and spectral distribution o f auto fluorescence seem to be the same for h u m a n and animal bone (Spas• and Weber, 1971). In our experiments the
D. Dekani6 et al. : Bone Fluorescence in Rats - Age and Sex Relationship Table 2.
79
Fluorescence intensity of powdered femoral bones of male and female rats of different age after one i. p. injection of 100 mg/kg OTC Fluorescence intensity (arbitrary units) Age (months)
Male Female
2 (13) y (14) *
4 (12) (10)
8 (19)(17)
12 (17) (15)
16 (19) (22)
20 (5) (10)
455 +_ 25a+ 314 _+ 11"
315 ___ 7,d 283 + 10 d
238 _+ 58 212 + 5 c
216 _+ 5~ s 192 _+ 4 Ns
214 _+ 4~ s 195 q- 4 b
233 _+ 8Ns 225 _+ 11
Values are means and S.E. Numbers in parentheses are numbers of animals: y males, x females. , - d Statistically significant differences between successional age groups at 5, 2, 1 and 0.1 ~ level respectively. w Statistically significant differences between sexes for corresponding age groups at 2, 1 and 0.1 level respectively NS = not significant
70
x.X'X" X
,7
.,~
io~.~ xX, x.x" [
,,~. "~
i
~
L
i
I
i
i
i
~, x L
.J
W o v e - I.ength ( n m )
Fig. 1. Characteristic spectral curves of OTC induced fluorescence and autofluorescence in rat femoral bone (powdered and intact) in relation to age and sex. Curve 1 - a l l untreated animals (powdered and intact femora) and all OTC treated adult females (intact femora). Curve 2 - all OTC treated young males and females (powdered and intact femora) and some OTC treated adult males (intact femora). Curves 3 and 4 - all OTC treated adult males and females (powdered femora) and majority of OTC treated adult males (intact femora) 70
III
SO
C Q o C
/
\ ;'x
~30
\
Q k. O
u.
",
10
tOO Wave
Fig. 2.
- tength
SO0 { nrn )
Spectral curves for gelatin-collagen (1) and OTC (2)
autofluorescence of rat bone was seen to increase with age confirming the observations of Prentice (1965) who, with a microscopic technique observed a more intensive fluorescence in older bones. Later Armstrong et al. (1972) quantitatively confirmed a significant increase of autofluorescence in human bone with age. However, the differences with ageing obserw~d in our study on rats seem to be much smaller (about 30 ~o) than the human data (about 100 ~). Further our results indicate no sex related differences in autofluorescence intensity. The observation of a decrease in the intensity of OTC induced fluorescence with age and a higher retention of the antibiotic in male than in female rats is in agreement with some previous results. As previously mentioned Milch et al. (1958) reported that young and adult bone could be differentiated on the basis of the intensity of the emitted light as well as its localization. Later on while introducing chemical methods for the quantitative estimation of tetracyclines in the bone tissue, Hawkins (1972) observed (on two age groups of animals only) that young rats retained more tetracycline than adult. Buyske et al. (1960) following the time course of tetracycline retention in young rats noticed that male rats retained more tetracycline in bones than age matched females. Although the mechanism and site of tetracycline deposition in calcified tissues are still uncertain (Harris et al., 1962; Urist and Ibsen, 1963; Steendijk, 1964) there is no doubt that in younger animals the fixation of antibiotics in bone is largely confined to grewing surfaces (Milch et al., 1958; Harris et al., 1962). Therefore a progressive fall in bone retention of OTC with advancing age as well as a higher deposition in males than in females could reflect the changes in growth rate accompanying age and sex. In adult bone the mineral and matrix turnover is reduced which is consistent with a lower OTC retention. The increase in the intensity of OTC induced fluorescence in the oldest animals as compared to adults, which has not yet been
80
reported by other authors, could be interpreted by a higher remodelling activity which opposes structural changes in aging bone (Jowsey, 1960; LaCroix and Coutelier, 1973). When interpreting the results of the OTC retention in bone it should also be borne in mind that they might be influenced by the rate of retention and elimination of the antibiotic from other body compartments. In a preliminary study we noticed a higher OTC concentration in the plasma in male rats than in female 4 - 2 4 h after a single intraperitoneal application (Dekani6, t975). This may indirectly cause an increased bone retention in males. This possibility has not been considered by other authors when discussing the differences in tetracycline retention in bone. Our spectrofluorometric studies of intact bone indicate a lowering of the OTC retention at the periosteal surface with increasing age. Epker and Frost (1966) obtained similar results in humans using a different experimental technique. It is interesting to note that adult females showed no signs of OTC retention at the periosteal surface. This might be due to the fact that at this age (12 months) female rats cease growing and their bones are not yet affected by the ageing processes which as shown previously in our strain of rats begin after the age of 16 months (Blanu~a, 1974). In conclusion-our results indicate that the determination of OTC retention in bone using our quantitative fluorometric method and the detection of OTC deposition at bone surface using a spectrofluorometric method might be a sensitive tool by which physiological changes in bone tissue related to age and sex may be detected in a simple, rapid and economic way. We believe that these methods might also be successfuly used in experimental studies of osteoporotic changes in bone. Further work along this line is in progress. Acknowledgement. The authors thank Miss Nada Breber for her valuable technical assistance.
REFERENCES Argauer, R. J., White, C. E. : Fluorescent compounds for calibration of excitation and emission units of spectroltuorometer. Anal. Chem. 36, 368-371 (1964) Armstrong, W. G., Horsley, H.J.: Isolation and porperties of fluorescent components associated with calcified tissue collagen. Calcif. Tiss. Res. 8, 197-210 (1972)
Pflfigers Arch. 370 (1977) Armstrong, W. G., Erholtz, L., Prentice, A. I. D. : Age-related accumulation in calcified connective tissues of UV absorbing material masking an increased collagen-bound fluorescence found in older tissues. IVth International Osteological Symposium, Abstract 4, Prague 1972 Bacbman, C. H., Ellis, E. H. : Fluorescence of bone. Nature 206, 1328-1331 (1965) Blanu~a, M.: Utjecaj nekih faktora na kinetiku metabolizma kalcija (PhD Thesis), University Zagreb 1974 Buyske, D. A., Eisner, H. J., Kelly, R. G. : Concentration and persistence of tetracycline and chlortetracycline in bone. J. Pharmacol. Exp. Ther. 130, 150-156 (1960) Dekani~, D. : Odredjivanje tetraciklinskih antibiotika u kostima (MSc Thesis), University of Zagreb 1971 Dekani+, D., Blanu~a, M., Kostial, K., Weber, K. : Odredjivanje tetraciklinskih antibiotika u ko~tanom tkivu i zubima mjerenjem fluorescencije. In: Antibiotici i antibiotska terapija (J. Fali~evac et al., eds.), pp. 364- 366. Zagreb: Ognjen Prica 1972 Dekani6, D. : Primjena tetraciklinskih antibiotika za studij metabolizma kalcija (PhD Thesis), University of Zagreb 1975 Epker, B. N., Frost, H. M. : Periosteal appositional bone growth from age two to age seventhy in man. A tetracycline evaluation. Anat. Rec. 154, 573-577 (1966) Frost, H. M. : Tetracycline-based histological analysis of bone remodeling. Calcif. Tiss. Res. 3, 211-237 (1969) Harris, W. H., Jackson, R. H., Jowsey, J. : The in vivo distribution of tetracyclines in canine bone. J. Bone Joint Surg. Am. 44, 1308-1320 (1962) Hawkins, K. I.: Fluorometric determination of demethylchlortetracycline and tetracycline in mammalian bone. Anal. Biochem. 45, 128-136 (1972) Himel, C.M., Mayer, R. T. : 5-Dimethylaminonaphtalene-l-suIfonic acid (DANS acid) as standard for quantum yield of fluorescence. Anal. Chem. 42, 130-132 (1970) Jowsey, J. : Age changes in human bone. Clin. Orthop. 17, 210 -218 (1960) LaCroix, P., Coutelier, L. : Microscopic de fluorescence et microradiographic dans l'6tude du vieillissement de l'os. In: Orthopaedic Surgery and Traumatology (J. Delchef, et al., eds.), pp. 361 - 363. Amsterdam: Excerpta Medica 1973 Lippert, E., N~igele, W., Seibold-Blankenstein, I., Staiger, U., Voss, W. : Messung von Fluorescenzspektren mit Hilfe yon Spectralpbotometern und Vergleicbsstandards. Z. anal. Chem. 170, 1 - 18 (1959) Milch, R. A., Rall, D. P., Tobie, J. E. : Fluorescence of tetracycline antibiotics in bone. J. Bone Joint Surg. Am. 40, 897 - 910 (1958) Prentice, A. I. D.: Bone autofluorescence and mineral content. Nature 206, 1167 (1965) Spasi6, P., Weber, K.: f2ber die natfirliche Fluoreszenz der Knochen. Acta Pharm. Jug. 21, 151-158 (1971) Steendijk, R. : Studies on the mechanism of the fixation of the tetracyclines to bone. In: Bone and Tooth (H. J. J. Blackwood, ed.), pp. 49--63. Oxford: Pergamon Press 1964 Urist, M. R., Ibsen, K. H. : Chemical reactivity of mineralized tissue with oxytetracycline. Arch. Pathol. 76, 484-496 (1963)
Received March 3, 1977
Pfliigers Arch. 370, 77-80 (1977)
EuropeanJoumal
of~y
9 by Springer-Verlag1977
Determination of Oxytetracycline Induced Fluorescence and Autofluorescence as an Indicator of Age and Sex Differences in Rat Femur* D A R I N K A D E K A N I C , K A R L O WEBER, and K R I S T A K O S T I A L Institute for Medical Research and Occupational Health, Mo~e Pijade 158, YU-41000 Zagreb, Yugoslavia
Summary. Simple and rapid methods for the determination and detection of autofluorescence and oxytetracycline (OTC) induced fluorescence in powdered and intact bone (without previous chemical extraction of the antibiotic) were used to estimate age and sex related differences in the rat femur. The autofluorescence always had collagen characteristics; it increased with age and was not sex dependent. The intensity of O T C induced fluorescence decreased with age, being higher in males than in females. In the oldest animals, however a slight increase in the fluorescence intensity was observed, and sex differences disappeared. The spectrofluorometric measurements of intact femora indicate a higher O T C retention on the bone surface of young animals and adult males but not in adult females. The results indicate that the determination of O T C retention in the bone using our quantitative fluorometric method and the detection of O T C deposition at bone surface using a spectrofluorometric technique can be a sensitive and inexpensive tool for experimental studies of bone changes in physiological and pathological conditions.
sensitive indicators of bone changes especially in osteoporosis. In 1958 Milch and collaborators stated in one of the earliest reports on tetracycline induced fluorescence in bone that with age changes in bone tissue can be detected grossly using this technique. Detailed investigations in this field were not performed, possibly because of the difficulties involved in quantitative estimation of tetracyclines in calcified tissues. Most authors over this period used tetracyclines only as a label for studying bone formation (Frost, 1969). The rare reports on bone autofluorescence showed that this type of bone fluorescence is also age related. These data indicate that autofluorescence and tetracycline induced fluorescence might be used for studying age related changes in the bone tissue. In the present investigation, which involves the use of simple and rapid methods for both the detection and measurement of autofluorescence and oxytetracycline (OTC) induced fluorescence in powdered and intact bone (without previous chemical extraction of the antibiotic), age and sex related differences in rat femur were studied.
K e y words: Bone - O T C induced fluorescence and autofluorescence - Intensity and spectrofluorometry Age and sex relationship.
METHODS Experimental Animals. Experiments were performed on male and female albino rats from own laboratory stock. The intensity of fluorescence was determined in powdered bone samples in groups of animals aged 2-20 months, each consisting of 5-22 rats. The spectral distribution of fluorescence of powdered as well as intact femoral bone was determined in the youngest-2 month old, and in adult-12 month old animals, divided into groups of 5-19 rats each.
-
INTRODUCTION Over the last 20 years several new methods have been introduced with the aim of developing more * This study was supported in part by the "Pliva", Pharmaceutical Works, Zagreb, Yugoslavia and US Department of Agriculture, Beltsville, Maryland Send offprints requests to K. Kostial at the above address
Oxytetracyeline Treatment. The treated rats received either one intraperitoneal injection of 100 mg OTC (Geomycin, "Pliva", Zagreb) per kilogram body weight (when the intensity of fluorescence was measured) or six intraperitoneal injections of 100 mg OTC per kilogram body weight (when the spectral distribution of fluorescence was determined). Control animals were given corresponding injections of a physiologicalsaline solution (0.9 % NaC1). The animals were killed by ether anaesthesia 72 h after the last injection.
78
Pflfigers Arch. 370 (1977)
Table 1. Autofluorescence intensity of powdered femoral bones of male and female rats of different age Fluorescence intensity (arbitrary units) Age (months)
Male Female
2 6 12 (10)y (20)x (10) (10) (10)(10)
20 (10) (19)
140_+ 3 c 140_+2 d
182• 184• 3
149_+2 d 170• 3 d 147_+2 d 168_+ 3 d
Values are means and S.E. Numbers in parentheses are numbers of animals: ymales xfemales ~,d Statistically significant differences between successional age groups at 1 and 0.1 ~ level respectively
Treatment of Bone Samples. Both femora were removed and cleaned from the connective tissues. The foramen nutricium was enlarged by means of a hand borer to a diameter of 1 - 2 mm and the bone marrow was removed by centrifngation (3000 g for 30 min). The bones were dried for 15 days in the dark at room temperature and after that both femora were ground into a fine powder with a mortar. We have shown previously that under such conditions consistent results can be obtained (Dekani6, 1971). For the determination of the spectral curves of intact (unpowdered) bone the right femur was also used after being dried for 15 days at room temperature. Measurement of Fluorescent Intensity and Its Spectral Distribution. The intensity of fluorescence of powdered bone samples was measured with a photoelectric fluorometer of own construction. The light source was a mercury lamp with appropriate filters to give maximum transmission at 366 nm. Each measurement was made at 45~ to the incident beam by comparison with a standard (filter paper coated with optical white). The precision of the method was _+2 ~. In our previous study we observed a linear relationship between the dose of intraperitoneally injected OTC (dose range 12.5- 200 mg/kg) and fluorescent intensity of powdered bone samples (Dekani6 et al., 1972). The spectral distribution of fluorescence in powdered and intact bone samples was determined with a commercial spectroftuorometer (Perkin Elmer-Hitachi MPF 2A). The same light source and angle of measurement were used as in the measurement of the intensity. The spectral distribution from quinine sulphate and 3-aminophthalimide in 0.1 N sulphuric acid served as standard (Lippert et al., 1959; Argauer and White, 1964; Himel and Mayer, 1970).
RESULTS
Intensity o f Autofluorescence and 0 TC Induced Fluorescence in Powdered Bone Autofluorescence. Table 1 shows the relative intensity o f autofluorescence in p o w d e r e d bone in relation to age and sex. The results show an almost linear relationship between auto fluorescence intensity and age. Samples f r o m y o u n g e r rats always h a d a significantly lower fluorescence intensity than those f r o m older animals while within the same age g r o u p the difference between male and female animals was n o t significant.
O T C Induced Fluorescence. Table 2 shows the relative intensity o f fluorescence o f p o w d e r e d f e m o r a
after a single i.p. injection o f OTC. The intensity was significantly higher in samples f r o m y o u n g animals than f r o m the older animals o f either sex until 12 m o n t h s of age. F r o m then onwards there was no further, change in either male or female animals. A statistically higher fluorescence intensity was generally observed in males. In the oldest animals (20 months) the intensity o f fluorescence was again slightly increased (statistically significant only in females) so that sex differences in b o n e fluorescence levels disappeared.
Spectrofluorometric Measurements of Autofluorescence and 0 TC Induced Fluorescence Powdered Bone Samples. Spectrofluorometric measurements resulted in curves o f four basic shapes: curves 1, 2, 3 and 4 as shown in Figure 1. Figure 2 shows the curves obtained for a commercial preparation (Gelatin W o r k s Ljubljana) o f gelatin-collagen (curve 1) and O T C (curve 2). P o w d e r e d bone samples f r o m untreated animals always gave curves o f the type for collagen fluorescence (Figs. I and 2, curve 1), while y o u n g O T C treated animals provided curves of the O T C type (Figs. 1 and 2, curve 2). Measurements f r o m p o w d e r e d f e m o r a o f adult O T C treated rats usually resulted in curves o f the type shown in Figure 1 curves 3 and 4 which resulted f r o m b o t h types of fluorescence (autofluorescence and O T C induced fluorescence).
Intact Femora. Intact (unpowdered) bone samples f r o m untreated animals again showed only the spectral characteristics o f collagen (Fig. 1, curve 1). The f e m o r a o f y o u n g O T C treated rats o f both sexes showed only the O T C characteristics (Fig. 1, curve 2). In adult O T C treated animals female f e m o r a showed no signs o f O T C induced fluorescence (Fig. 1, curve 1) indicating no O T C deposition at periosteal surface. M o s t male femora showed curves which resulted f r o m b o t h types o f fluorescence (curves 3 and 4 in Fig. 1) and sometimes even only the O T C spectral curve (Fig. 1, curve 2). DISCUSSION O u r results which concern the autofluorescence of the rat bone agree in general with the few data available in literature. The spectrum o f the emitted light has collagen characteristics, as in b o n e samples f r o m h u m a n s ( B a c h m a n and Ellis, 1965; A r m s t r o n g and Horsley, 1972), confirming an earlier report that the colour and spectral distribution o f auto fluorescence seem to be the same for h u m a n and animal bone (Spas• and Weber, 1971). In our experiments the
D. Dekani6 et al. : Bone Fluorescence in Rats - Age and Sex Relationship Table 2.
79
Fluorescence intensity of powdered femoral bones of male and female rats of different age after one i. p. injection of 100 mg/kg OTC Fluorescence intensity (arbitrary units) Age (months)
Male Female
2 (13) y (14) *
4 (12) (10)
8 (19)(17)
12 (17) (15)
16 (19) (22)
20 (5) (10)
455 +_ 25a+ 314 _+ 11"
315 ___ 7,d 283 + 10 d
238 _+ 58 212 + 5 c
216 _+ 5~ s 192 _+ 4 Ns
214 _+ 4~ s 195 q- 4 b
233 _+ 8Ns 225 _+ 11
Values are means and S.E. Numbers in parentheses are numbers of animals: y males, x females. , - d Statistically significant differences between successional age groups at 5, 2, 1 and 0.1 ~ level respectively. w Statistically significant differences between sexes for corresponding age groups at 2, 1 and 0.1 level respectively NS = not significant
70
x.X'X" X
,7
.,~
io~.~ xX, x.x" [
,,~. "~
i
~
L
i
I
i
i
i
~, x L
.J
W o v e - I.ength ( n m )
Fig. 1. Characteristic spectral curves of OTC induced fluorescence and autofluorescence in rat femoral bone (powdered and intact) in relation to age and sex. Curve 1 - a l l untreated animals (powdered and intact femora) and all OTC treated adult females (intact femora). Curve 2 - all OTC treated young males and females (powdered and intact femora) and some OTC treated adult males (intact femora). Curves 3 and 4 - all OTC treated adult males and females (powdered femora) and majority of OTC treated adult males (intact femora) 70
III
SO
C Q o C
/
\ ;'x
~30
\
Q k. O
u.
",
10
tOO Wave
Fig. 2.
- tength
SO0 { nrn )
Spectral curves for gelatin-collagen (1) and OTC (2)
autofluorescence of rat bone was seen to increase with age confirming the observations of Prentice (1965) who, with a microscopic technique observed a more intensive fluorescence in older bones. Later Armstrong et al. (1972) quantitatively confirmed a significant increase of autofluorescence in human bone with age. However, the differences with ageing obserw~d in our study on rats seem to be much smaller (about 30 ~o) than the human data (about 100 ~). Further our results indicate no sex related differences in autofluorescence intensity. The observation of a decrease in the intensity of OTC induced fluorescence with age and a higher retention of the antibiotic in male than in female rats is in agreement with some previous results. As previously mentioned Milch et al. (1958) reported that young and adult bone could be differentiated on the basis of the intensity of the emitted light as well as its localization. Later on while introducing chemical methods for the quantitative estimation of tetracyclines in the bone tissue, Hawkins (1972) observed (on two age groups of animals only) that young rats retained more tetracycline than adult. Buyske et al. (1960) following the time course of tetracycline retention in young rats noticed that male rats retained more tetracycline in bones than age matched females. Although the mechanism and site of tetracycline deposition in calcified tissues are still uncertain (Harris et al., 1962; Urist and Ibsen, 1963; Steendijk, 1964) there is no doubt that in younger animals the fixation of antibiotics in bone is largely confined to grewing surfaces (Milch et al., 1958; Harris et al., 1962). Therefore a progressive fall in bone retention of OTC with advancing age as well as a higher deposition in males than in females could reflect the changes in growth rate accompanying age and sex. In adult bone the mineral and matrix turnover is reduced which is consistent with a lower OTC retention. The increase in the intensity of OTC induced fluorescence in the oldest animals as compared to adults, which has not yet been
80
reported by other authors, could be interpreted by a higher remodelling activity which opposes structural changes in aging bone (Jowsey, 1960; LaCroix and Coutelier, 1973). When interpreting the results of the OTC retention in bone it should also be borne in mind that they might be influenced by the rate of retention and elimination of the antibiotic from other body compartments. In a preliminary study we noticed a higher OTC concentration in the plasma in male rats than in female 4 - 2 4 h after a single intraperitoneal application (Dekani6, t975). This may indirectly cause an increased bone retention in males. This possibility has not been considered by other authors when discussing the differences in tetracycline retention in bone. Our spectrofluorometric studies of intact bone indicate a lowering of the OTC retention at the periosteal surface with increasing age. Epker and Frost (1966) obtained similar results in humans using a different experimental technique. It is interesting to note that adult females showed no signs of OTC retention at the periosteal surface. This might be due to the fact that at this age (12 months) female rats cease growing and their bones are not yet affected by the ageing processes which as shown previously in our strain of rats begin after the age of 16 months (Blanu~a, 1974). In conclusion-our results indicate that the determination of OTC retention in bone using our quantitative fluorometric method and the detection of OTC deposition at bone surface using a spectrofluorometric method might be a sensitive tool by which physiological changes in bone tissue related to age and sex may be detected in a simple, rapid and economic way. We believe that these methods might also be successfuly used in experimental studies of osteoporotic changes in bone. Further work along this line is in progress. Acknowledgement. The authors thank Miss Nada Breber for her valuable technical assistance.
REFERENCES Argauer, R. J., White, C. E. : Fluorescent compounds for calibration of excitation and emission units of spectroltuorometer. Anal. Chem. 36, 368-371 (1964) Armstrong, W. G., Horsley, H.J.: Isolation and porperties of fluorescent components associated with calcified tissue collagen. Calcif. Tiss. Res. 8, 197-210 (1972)
Pflfigers Arch. 370 (1977) Armstrong, W. G., Erholtz, L., Prentice, A. I. D. : Age-related accumulation in calcified connective tissues of UV absorbing material masking an increased collagen-bound fluorescence found in older tissues. IVth International Osteological Symposium, Abstract 4, Prague 1972 Bacbman, C. H., Ellis, E. H. : Fluorescence of bone. Nature 206, 1328-1331 (1965) Blanu~a, M.: Utjecaj nekih faktora na kinetiku metabolizma kalcija (PhD Thesis), University Zagreb 1974 Buyske, D. A., Eisner, H. J., Kelly, R. G. : Concentration and persistence of tetracycline and chlortetracycline in bone. J. Pharmacol. Exp. Ther. 130, 150-156 (1960) Dekani~, D. : Odredjivanje tetraciklinskih antibiotika u kostima (MSc Thesis), University of Zagreb 1971 Dekani+, D., Blanu~a, M., Kostial, K., Weber, K. : Odredjivanje tetraciklinskih antibiotika u ko~tanom tkivu i zubima mjerenjem fluorescencije. In: Antibiotici i antibiotska terapija (J. Fali~evac et al., eds.), pp. 364- 366. Zagreb: Ognjen Prica 1972 Dekani6, D. : Primjena tetraciklinskih antibiotika za studij metabolizma kalcija (PhD Thesis), University of Zagreb 1975 Epker, B. N., Frost, H. M. : Periosteal appositional bone growth from age two to age seventhy in man. A tetracycline evaluation. Anat. Rec. 154, 573-577 (1966) Frost, H. M. : Tetracycline-based histological analysis of bone remodeling. Calcif. Tiss. Res. 3, 211-237 (1969) Harris, W. H., Jackson, R. H., Jowsey, J. : The in vivo distribution of tetracyclines in canine bone. J. Bone Joint Surg. Am. 44, 1308-1320 (1962) Hawkins, K. I.: Fluorometric determination of demethylchlortetracycline and tetracycline in mammalian bone. Anal. Biochem. 45, 128-136 (1972) Himel, C.M., Mayer, R. T. : 5-Dimethylaminonaphtalene-l-suIfonic acid (DANS acid) as standard for quantum yield of fluorescence. Anal. Chem. 42, 130-132 (1970) Jowsey, J. : Age changes in human bone. Clin. Orthop. 17, 210 -218 (1960) LaCroix, P., Coutelier, L. : Microscopic de fluorescence et microradiographic dans l'6tude du vieillissement de l'os. In: Orthopaedic Surgery and Traumatology (J. Delchef, et al., eds.), pp. 361 - 363. Amsterdam: Excerpta Medica 1973 Lippert, E., N~igele, W., Seibold-Blankenstein, I., Staiger, U., Voss, W. : Messung von Fluorescenzspektren mit Hilfe yon Spectralpbotometern und Vergleicbsstandards. Z. anal. Chem. 170, 1 - 18 (1959) Milch, R. A., Rall, D. P., Tobie, J. E. : Fluorescence of tetracycline antibiotics in bone. J. Bone Joint Surg. Am. 40, 897 - 910 (1958) Prentice, A. I. D.: Bone autofluorescence and mineral content. Nature 206, 1167 (1965) Spasi6, P., Weber, K.: f2ber die natfirliche Fluoreszenz der Knochen. Acta Pharm. Jug. 21, 151-158 (1971) Steendijk, R. : Studies on the mechanism of the fixation of the tetracyclines to bone. In: Bone and Tooth (H. J. J. Blackwood, ed.), pp. 49--63. Oxford: Pergamon Press 1964 Urist, M. R., Ibsen, K. H. : Chemical reactivity of mineralized tissue with oxytetracycline. Arch. Pathol. 76, 484-496 (1963)
Received March 3, 1977
