Archives of
RDesermato.logical
Arch. Dermatol. Res. 263, 135-138 (1978)
earcn 9 Springer-Verlag 1978
Effects of Tetracycline on the Absorption of 65Zinc in Rats Kaare Weismann, Lone Knudsen, and Henrik H~byer Department of Dermatology, University of Copenhagen, Rigshospital, DK-2100 Copenhagen, Denmark
Summary. The effects of tetracycline on the absorption of orally ingested 6SZn was studied on rats by whole body counting assay. 65Zn was given as a single dose to groups of rats, five in each, which were started on tetracycline, Tetracycline was given in daily doses of 25 mg, 50 rag, 100 rag, and 200 mg for 10 days. From the 6th day of the study, 65Zn retention when plotted against time in a semilog plot, approximated linearity. The net absorption of 65Zn in the various groups was determined by extrapolating to zero time the linear curve segment of individual retention curves. Except for the lowest tetracycline dosage, tetracycline significantly impaired 65Zn absorption. The elimination rate of retained 65Zn from the 6th day was significantly higher in the group receiving 200 mg tetracycline as compared with the control group.
Key words: Tetracycline - Whole body counting - 65Zn. Zusammenfassung.Der EinfluB von Tetrazyklin auf die Absorption von oral verabreichtem 65Zn wurde bei Ratten im Ganzk6rperzfihler untersucht. 65Zn wurde in einer einzelnen Dosis den verschiedenen Tiergruppen gegeben. Die tats/ichliche Dosis des Tetrazyklins war 25 mg, 50 mg, 100 mg und 200 mg tiber 10 Tage. Vom 6. Tage an zeigte die Zn-Retention in semilogarithmischer Darstellung in etwa einen linearen Verlauf. Die 65Zn-Absorption wurde in den verschiedenen Gruppen dutch Extrapolierung zur 0-Zeit der linearen Kurvensegmente mit individuell variabler Retention durchgeftihrt. Mit Ausnahme der 25 mg Tetrazyklin-Dosis wurde bei h6herer Tetrazyklindosis eine signifikante Absorptionshemmung yon 65Zn festgestellt. Die Eliminationsrate yon reteniertern 65Zn war vom 6. Tage signifikant h6her bei der 200 mg Tetrazyklingabe im Vergleich mit der Kontrollgruppe. Sehliisselwiirter: Tetrazyklin - Ganzk6rperz/ihlung - 65Zn.
Offprint requests to: Dr. K. Weismann (address see above) 0340-3696/78/0263/0135/$1.00
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The a b s o r p t i o n o f orally ingested tetracycline (TC) is i m p a i r e d b y the a d m i n i s t r a t i o n o f t r i - a n d b i v a l e n t metals, including zinc [1, 3, 5]. A s T C f o r m s chelates with d i v a l e n t c a t i o n s in 1 : 1 r a t i o a t low p H a n d 2:1 at high p H [3], it can be expected t h a t the intestinal zinc u p t a k e is decreased d u r i n g T C t h e r a p y . This effect o f TC, however, has n o t been d e m o n s t r a t e d experimentally. T h e p r e s e n t s t u d y was designed to e v a l u a t e the interference o f T C with zinc a b s o r p t i o n b y m e a n s o f w h o l e b o d y c o u n t i n g technique a p p l i e d to rats.
Material and Methods Twenty-five male albino rats with an average weight of 150 g were assigned to follr experimental groups receiving TC and to one control group, five rats in each. The animals were kept in plastic cages and were allowed free access throughout the study to a diet containing 43 gg zinc/g and I mg ealcium/g. Each rat received a single dose of 0.5 gCi 6SZn by gavage (carrier-free 65ZNC12 (code ZAS 2), The Radiochemical Centre Ltd Amersham, England). The doses were made up to one ml by isotone NaCI water. Immediately after ~SZn ingestion, the experimental groups were started on TC therapy, administered by gavage. The groups received TC doses of 25 mg, 50 rag, 100rag, and 200rag daily for 10 days. TC was prepared as emulsions. 6~Zn activity was measured by a whole animal detector (Armac440, Packards Instruments Company Inc., La-Grange, Illinois, U.S.A.). The initial measurement was performed about 1h after garage of 6SZn, and the retention of the isotope, expressed as per cent of the administered dose, was followed during ten days' TC therapy. Countings were corrected for background activity, whereas 65Zn decay was disregarded due to its half-life of 245 days. A 65Zn standard was counted on each counting day to ensure counter stability. The net absorption of 65Zn was determined as described by Heth and Hoekstra [21 by plotting per cent retained 65Zn against time in a semilog plot and extrapolating to zero time the approximatelylinear curve segment, appearing after the bulk of 65Zn had been excreted via the feces. The elimination rate of 6SZn from day 6 to day 10 was estimated from the slope of the retention curves, as determined by least squares regression analysis. It was expressed as per cent decrease per day of retained 6SZn. Statistical analysis was performed by means of the t-test according to Dunnett's method, using a pooled variance estimate [4]. The relation between the drug doses and the 6SZnnet absorption was tested by polynomial regression analysis.
Results T h e 6SZn r e t e n t i o n curves o f all g r o u p s a p p r o x i m a t e d linearity f r o m d a y 6 to d a y 10, a n d e x t r a p o l a t i o n to zero time was p e r f o r m e d in each a n i m a l to o b t a i n the 6SZn net a b s o r p t i o n value (Fig. 1, T a b l e 1). T h e r e was a t e n d e n c y t o w a r d s l o w e r 65Zn net a b s o r p t i o n with increasing T C doses, b u t the c o r r e l a t i o n was n o t statistically significant (0.1 > P > 0.05). T h e ~SZn net a b s o r p t i o n was significantly lower in the g r o u p s receiving 50 mg, 100 mg, a n d 200 m g T C daily as c o m p a r e d with the c o n t r o l s (Table 1). T h e p e r cent decrease p e r d a y o f r e t a i n e d 65Zn was significantly higher in the 200 m g T C g r o u p c o m p a r e d w i t h the c o n t r o l g r o u p , as d e t e r m i n e d f r o m the 6th to the 10th d a y o f the s t u d y ( T a b l e 1). In the c o n t r o l g r o u p which was f o l l o w e d for 30 days, a less sloping segment o f the 65Zn r e t e n t i o n curves a p p e a r e d a b o u t 2 weeks after a d m i n i s t r a t i o n o f 65Zn" T h e p e r cent decrease p e r d a y o f r e t a i n e d 65Zn f r o m a b o u t 2 w e e k s a n d o n w a r d s was
Tetracycline and 6SZn Absorption
137
120
6O
Na N 40
~.20
CONTROL ~
20
~
~
TETRACYCLINE 25 , ,
~
TEFRACYO[I~E N mg FEFRAOYOLINE]00 mg TETRACYCLINE200 mg
i
i
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i
2
3
4
5
D
7
8
9
lO
DAYS AFTER 65Zn ADMINISTRATION
Fig. 1. Retention curves of orally ingested 6SZn in rats. The 6SZn is given as a single dose on day 0 when the groups are started on tetracycline. The tetracycline doses are given daily up to the 10th day after 6SZn administration. Each point is the mean value of five rats. On day 3 and on days 6 through 10, 65Zn retention of the groups receiving 50 mg, 100rag, and 200mg tetracycline is significantly lower as compared with the control group (P < 0.05 on day 3, else P < 0.01)
Table. 1. Extrapolated net absoption of 65Zn and elimination rate of retained 65Zn from day 6 to day 10 in tetracycline treated rats. Values are given as mean +_ standard deviation (SD.). N u m b e r of rats in each group = 5 Experimental dosage
65Zn net absorption (%)
Decrease per day of retained 6~Zn (%)
Tetracycline 25 mg/day Tetracycline 50 rag/day Tetracycline 100 rag/day Tetracycline 200 rag/day Control group
33.8 • 5.2 27.0 4-_3.1 a 25.2 _+ 3.7 u 25.6 _+ 3.7 b 37.6 + 8.7
3.6 3.6 3.7 5.5 3.3
+_ 0.5 + 0.3 _+0.2 _+0.5 r + 0.3
~,b significantly decreased as compared to the control group, P < 0.05 and P < 0.01, respectively. ~ significantly increased as compared to the control group, P < 0.01
2.7 + 0.05, which is significantly lower than the elimination rate from day 6 to day l0 (cf. Table 1, P < 0.01, t-test for paired data).
Discussion
The present results show that high oral TC doses impair the absorption of a single 65Zn dose in rats. The lowest experimental TC dose of 25mg daily, which corresponds to about 170mg/kg/day, did not decrease 65Zn net absorption significantly, As this dosage is far higher than used for man, ranging from about 5 25 mg/kg/day, the significance of the findings to human medicine seems obscure. However, on the basis of this experiment we can not exclude that TC therapy might interfere with the zinc uptake in persons having a marginal zinc intake and being in
138
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need for extra zinc for growth and tissue restorement. Furthermore, the zinc bioavailability and mineral content of the rat fodder probably differs from human diets. This might very well play a role for the chelating properties of TC. The whole body counting assay has the disadvantage of providing no details about where in the body 65Zn is present, and we do not know if it is freely available or firmly bound to TC. The increased elimination rate of retained 65Zn in the rats receiving 200 mg TC daily might indicate loss of TC-bound 6SZn via the urine or it might be explained by trapping recirculating 65Zn in the intestine by orally ingested TC. The present experiment provides no information on the effects of prolonged TC therapy on zinc absorption, and the effects of single TC doses also remain to be studied. Finally, it should be mentioned that the third component of the retention curves observed in the control group might also be influenced by administration of TC. The appearance of this curve segment shows us that the part of the retention curves used for extrapolation can not be strictly linear although it appears so from the graphs. Acknowledgments.We wish to thank the Department of Nuclear Medicine NU, Rigshospital, for kindly letting us use the Armac detector. Tetracycline emulsions were supplied by the Pharmacy Department, Rigshospital. Statistical analyses were performed by licla, tech. Aa. V~lund.
References 1. Andersson, K. E., Bratt, L., Dencker, H., Kamme, C., Lanner, E.: Inhibition of tetracycline absorption by zinc. Europ. J. Clin. Pharmacol. 10, 5 9 - 6 2 (1976) 2. Heth, D. A., Hoekstra, W. G. : Zinc-65 absorption and turnover in rats I. A procedure to determine zinc-65 absorption and the antagonistic effect of,calcium in a practical diet. J. Nutr. 85, 367-374 (1965) 3. Mapp, R. K., McCarthy, T. J. : The effect of zinc sulphate and of bicitropeptide on tetracycline absorption. S. Aft. Med. J. 50, 1829-1830 (1~)76) 4. Owen, D. B. : Handbook of Statistical Tables. Reading: Addison-Wesley Publishing Company 1962 5. Penttil~i, O., Hurme, H,, Neuvonen, P. J. : Effect of zinc sulphate on the absorption of tetracycline and doxycycline in man. Europ. J. Clin, PharmacoL 9, 131 - 1 3 4 (1975) Received June 16, 1978
RDesermato.logical
Arch. Dermatol. Res. 263, 135-138 (1978)
earcn 9 Springer-Verlag 1978
Effects of Tetracycline on the Absorption of 65Zinc in Rats Kaare Weismann, Lone Knudsen, and Henrik H~byer Department of Dermatology, University of Copenhagen, Rigshospital, DK-2100 Copenhagen, Denmark
Summary. The effects of tetracycline on the absorption of orally ingested 6SZn was studied on rats by whole body counting assay. 65Zn was given as a single dose to groups of rats, five in each, which were started on tetracycline, Tetracycline was given in daily doses of 25 mg, 50 rag, 100 rag, and 200 mg for 10 days. From the 6th day of the study, 65Zn retention when plotted against time in a semilog plot, approximated linearity. The net absorption of 65Zn in the various groups was determined by extrapolating to zero time the linear curve segment of individual retention curves. Except for the lowest tetracycline dosage, tetracycline significantly impaired 65Zn absorption. The elimination rate of retained 65Zn from the 6th day was significantly higher in the group receiving 200 mg tetracycline as compared with the control group.
Key words: Tetracycline - Whole body counting - 65Zn. Zusammenfassung.Der EinfluB von Tetrazyklin auf die Absorption von oral verabreichtem 65Zn wurde bei Ratten im Ganzk6rperzfihler untersucht. 65Zn wurde in einer einzelnen Dosis den verschiedenen Tiergruppen gegeben. Die tats/ichliche Dosis des Tetrazyklins war 25 mg, 50 mg, 100 mg und 200 mg tiber 10 Tage. Vom 6. Tage an zeigte die Zn-Retention in semilogarithmischer Darstellung in etwa einen linearen Verlauf. Die 65Zn-Absorption wurde in den verschiedenen Gruppen dutch Extrapolierung zur 0-Zeit der linearen Kurvensegmente mit individuell variabler Retention durchgeftihrt. Mit Ausnahme der 25 mg Tetrazyklin-Dosis wurde bei h6herer Tetrazyklindosis eine signifikante Absorptionshemmung yon 65Zn festgestellt. Die Eliminationsrate yon reteniertern 65Zn war vom 6. Tage signifikant h6her bei der 200 mg Tetrazyklingabe im Vergleich mit der Kontrollgruppe. Sehliisselwiirter: Tetrazyklin - Ganzk6rperz/ihlung - 65Zn.
Offprint requests to: Dr. K. Weismann (address see above) 0340-3696/78/0263/0135/$1.00
136
K. Weismann et al.
The a b s o r p t i o n o f orally ingested tetracycline (TC) is i m p a i r e d b y the a d m i n i s t r a t i o n o f t r i - a n d b i v a l e n t metals, including zinc [1, 3, 5]. A s T C f o r m s chelates with d i v a l e n t c a t i o n s in 1 : 1 r a t i o a t low p H a n d 2:1 at high p H [3], it can be expected t h a t the intestinal zinc u p t a k e is decreased d u r i n g T C t h e r a p y . This effect o f TC, however, has n o t been d e m o n s t r a t e d experimentally. T h e p r e s e n t s t u d y was designed to e v a l u a t e the interference o f T C with zinc a b s o r p t i o n b y m e a n s o f w h o l e b o d y c o u n t i n g technique a p p l i e d to rats.
Material and Methods Twenty-five male albino rats with an average weight of 150 g were assigned to follr experimental groups receiving TC and to one control group, five rats in each. The animals were kept in plastic cages and were allowed free access throughout the study to a diet containing 43 gg zinc/g and I mg ealcium/g. Each rat received a single dose of 0.5 gCi 6SZn by gavage (carrier-free 65ZNC12 (code ZAS 2), The Radiochemical Centre Ltd Amersham, England). The doses were made up to one ml by isotone NaCI water. Immediately after ~SZn ingestion, the experimental groups were started on TC therapy, administered by gavage. The groups received TC doses of 25 mg, 50 rag, 100rag, and 200rag daily for 10 days. TC was prepared as emulsions. 6~Zn activity was measured by a whole animal detector (Armac440, Packards Instruments Company Inc., La-Grange, Illinois, U.S.A.). The initial measurement was performed about 1h after garage of 6SZn, and the retention of the isotope, expressed as per cent of the administered dose, was followed during ten days' TC therapy. Countings were corrected for background activity, whereas 65Zn decay was disregarded due to its half-life of 245 days. A 65Zn standard was counted on each counting day to ensure counter stability. The net absorption of 65Zn was determined as described by Heth and Hoekstra [21 by plotting per cent retained 65Zn against time in a semilog plot and extrapolating to zero time the approximatelylinear curve segment, appearing after the bulk of 65Zn had been excreted via the feces. The elimination rate of 6SZn from day 6 to day 10 was estimated from the slope of the retention curves, as determined by least squares regression analysis. It was expressed as per cent decrease per day of retained 6SZn. Statistical analysis was performed by means of the t-test according to Dunnett's method, using a pooled variance estimate [4]. The relation between the drug doses and the 6SZnnet absorption was tested by polynomial regression analysis.
Results T h e 6SZn r e t e n t i o n curves o f all g r o u p s a p p r o x i m a t e d linearity f r o m d a y 6 to d a y 10, a n d e x t r a p o l a t i o n to zero time was p e r f o r m e d in each a n i m a l to o b t a i n the 6SZn net a b s o r p t i o n value (Fig. 1, T a b l e 1). T h e r e was a t e n d e n c y t o w a r d s l o w e r 65Zn net a b s o r p t i o n with increasing T C doses, b u t the c o r r e l a t i o n was n o t statistically significant (0.1 > P > 0.05). T h e ~SZn net a b s o r p t i o n was significantly lower in the g r o u p s receiving 50 mg, 100 mg, a n d 200 m g T C daily as c o m p a r e d with the c o n t r o l s (Table 1). T h e p e r cent decrease p e r d a y o f r e t a i n e d 65Zn was significantly higher in the 200 m g T C g r o u p c o m p a r e d w i t h the c o n t r o l g r o u p , as d e t e r m i n e d f r o m the 6th to the 10th d a y o f the s t u d y ( T a b l e 1). In the c o n t r o l g r o u p which was f o l l o w e d for 30 days, a less sloping segment o f the 65Zn r e t e n t i o n curves a p p e a r e d a b o u t 2 weeks after a d m i n i s t r a t i o n o f 65Zn" T h e p e r cent decrease p e r d a y o f r e t a i n e d 65Zn f r o m a b o u t 2 w e e k s a n d o n w a r d s was
Tetracycline and 6SZn Absorption
137
120
6O
Na N 40
~.20
CONTROL ~
20
~
~
TETRACYCLINE 25 , ,
~
TEFRACYO[I~E N mg FEFRAOYOLINE]00 mg TETRACYCLINE200 mg
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DAYS AFTER 65Zn ADMINISTRATION
Fig. 1. Retention curves of orally ingested 6SZn in rats. The 6SZn is given as a single dose on day 0 when the groups are started on tetracycline. The tetracycline doses are given daily up to the 10th day after 6SZn administration. Each point is the mean value of five rats. On day 3 and on days 6 through 10, 65Zn retention of the groups receiving 50 mg, 100rag, and 200mg tetracycline is significantly lower as compared with the control group (P < 0.05 on day 3, else P < 0.01)
Table. 1. Extrapolated net absoption of 65Zn and elimination rate of retained 65Zn from day 6 to day 10 in tetracycline treated rats. Values are given as mean +_ standard deviation (SD.). N u m b e r of rats in each group = 5 Experimental dosage
65Zn net absorption (%)
Decrease per day of retained 6~Zn (%)
Tetracycline 25 mg/day Tetracycline 50 rag/day Tetracycline 100 rag/day Tetracycline 200 rag/day Control group
33.8 • 5.2 27.0 4-_3.1 a 25.2 _+ 3.7 u 25.6 _+ 3.7 b 37.6 + 8.7
3.6 3.6 3.7 5.5 3.3
+_ 0.5 + 0.3 _+0.2 _+0.5 r + 0.3
~,b significantly decreased as compared to the control group, P < 0.05 and P < 0.01, respectively. ~ significantly increased as compared to the control group, P < 0.01
2.7 + 0.05, which is significantly lower than the elimination rate from day 6 to day l0 (cf. Table 1, P < 0.01, t-test for paired data).
Discussion
The present results show that high oral TC doses impair the absorption of a single 65Zn dose in rats. The lowest experimental TC dose of 25mg daily, which corresponds to about 170mg/kg/day, did not decrease 65Zn net absorption significantly, As this dosage is far higher than used for man, ranging from about 5 25 mg/kg/day, the significance of the findings to human medicine seems obscure. However, on the basis of this experiment we can not exclude that TC therapy might interfere with the zinc uptake in persons having a marginal zinc intake and being in
138
K. Weismann et al.
need for extra zinc for growth and tissue restorement. Furthermore, the zinc bioavailability and mineral content of the rat fodder probably differs from human diets. This might very well play a role for the chelating properties of TC. The whole body counting assay has the disadvantage of providing no details about where in the body 65Zn is present, and we do not know if it is freely available or firmly bound to TC. The increased elimination rate of retained 65Zn in the rats receiving 200 mg TC daily might indicate loss of TC-bound 6SZn via the urine or it might be explained by trapping recirculating 65Zn in the intestine by orally ingested TC. The present experiment provides no information on the effects of prolonged TC therapy on zinc absorption, and the effects of single TC doses also remain to be studied. Finally, it should be mentioned that the third component of the retention curves observed in the control group might also be influenced by administration of TC. The appearance of this curve segment shows us that the part of the retention curves used for extrapolation can not be strictly linear although it appears so from the graphs. Acknowledgments.We wish to thank the Department of Nuclear Medicine NU, Rigshospital, for kindly letting us use the Armac detector. Tetracycline emulsions were supplied by the Pharmacy Department, Rigshospital. Statistical analyses were performed by licla, tech. Aa. V~lund.
References 1. Andersson, K. E., Bratt, L., Dencker, H., Kamme, C., Lanner, E.: Inhibition of tetracycline absorption by zinc. Europ. J. Clin. Pharmacol. 10, 5 9 - 6 2 (1976) 2. Heth, D. A., Hoekstra, W. G. : Zinc-65 absorption and turnover in rats I. A procedure to determine zinc-65 absorption and the antagonistic effect of,calcium in a practical diet. J. Nutr. 85, 367-374 (1965) 3. Mapp, R. K., McCarthy, T. J. : The effect of zinc sulphate and of bicitropeptide on tetracycline absorption. S. Aft. Med. J. 50, 1829-1830 (1~)76) 4. Owen, D. B. : Handbook of Statistical Tables. Reading: Addison-Wesley Publishing Company 1962 5. Penttil~i, O., Hurme, H,, Neuvonen, P. J. : Effect of zinc sulphate on the absorption of tetracycline and doxycycline in man. Europ. J. Clin, PharmacoL 9, 131 - 1 3 4 (1975) Received June 16, 1978
