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Clearance of Sulfamethoxazole in Eggs and Tissues of Chickens
Clearance of Sulfamethoxazole in Eggs and Tissues of Chickens H. OIKAWA1, K. NAKAMOTO 2 , K. HIROTA 1 and K. KATAGIRI 1 'Aburahi Laboratories, Shionogi and Co., Ltd., Koka-cho, Shiga Prefecture, 520—34, Japan, and Plant and Animal Science Development, Shionogi and Co., Ltd., 3—12 Dosho-machi, Higashi-ku, Osaka, 541, Japan
2
(Received for publication August 30, 1976)
Poultry Science 5 6 : 8 1 3 - 8 2 1 , 1977
INTRODUCTION Sulfamethoxazole (SMX) or sulfisomezole, a kind of sulfa drug, is a c h e m o t h e r a p e u t i c agent being used for t h e t r e a t m e n t of p r o t o z o a l and bacterial diseases of animals, and has been extensively used for t r e a t m e n t of chicken coccidiosis and infectious coryza. Considerable work on t h e clearance of t h e sulfa drug in t h e edible tissue a n d eggs of chickens was p e r f o r m e d (Blom, 1 9 7 5 ; Fellig et al., 1971 a,b; Krieg, 1 9 6 6 ; Krieg et al, 1 9 7 6 ; Laurencot et al., 1 9 7 2 ; Liider et al., 1 9 7 4 ; Nakatani et al., 1 9 6 3 ; Onodera et al, 1 9 7 0 ; a n d Righter et al, 1970, 1 9 7 1 ) . More detailed information is needed. T h e present s t u d y was u n d e r t a k e n t o determine t h e clearance patterns of SMX in egg a l b u m e n , y o l k and tissues after withdrawal of t h e drug in chicken t o which t h e drug was administered in t h e feed or intramuscularly for 5 successive days.
2.0 kg. in weight were used in t h e trial on eggs. Broiler chickens of b o t h sexes aged 4 0 t o 6 0 days old a n d 1.5 t o 2 . 0 kg. in weight were used in t h e trial o n tissues. These chickens purchased at a p o u l t r y farm in t h e n e i g h b o r h o o d were i n t r o d u c e d i n t o t h e single cage in t h e animal house of this laborat o r y t w o w e e k s before t h e m e d i c a t i o n . N o disease was observed. Animal h o u s e was maintained at 25±2 C. in r o o m t e m p e r a t u r e and 45—65% in h u m i d i t y . F o o d and water were taken ad lib. F o o d for layers was s u p p l e m e n t e d with o y s t e r shells a n d vitamins. 2. Medication and sampling, a) Drugs used. "Sinomin p o w d e r for animal u s e " containing SMX free base p r e m i x e d with starch p o w d e r at a 10% n e t c o n c e n t r a t i o n , and " S i n o m i n injection for animal u s e " containing SMX-monoe t h a n o l a m i n e a t a n e t value of 1 0 0 m g . per ml. as SMX free base were used. Chemical s t r u c t u r e of SMX is as follows.
MATERIALS AND METHODS 1. Animals and rearing conditions. White Leghorn h e n s , 7 t o 10 m o n t h s old a n d 1.5 t o
H2IM-// 7—S02 \=Z/
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ABSTRACT Clearance of sulfamethoxazole (SMX, or sulfisomezole) in eggs and tissues of chicken after withdrawal of the drug which was medicated feed additively (at 0.2 or 0.4%) or administered intramuscularly (100 mg./kg., twice daily) for 5 successive days was determined using a fluorescamine reagent. Clearance patterns of SMX were found to be different between albumen and yolk. SMX level in albumen diminished linearly to below 0.1 p.p.m. (sensitivity level) by 5 days after the cessation of medication. On the other hand, SMX level in yolk remained at a plateau until 4 days, and thereafter decreased linearly to below 0.1 p.p.m. by 10 days. SMX levels in most tissues diminished below 0.1 p.p.m. by 3 days and those in kidney and skin by 4 days. Clearance patterns in tissue were quite similar to those in egg albumen. Proportion of acetylated-SMX to total-one in both albumen and yolk tended to increase up to 10% with the lapse of time after drug withdrawal. Acetylation was high in liver (36.5—43.6%), spleen skin and fat, but low in breast muscle and kidney (3.7—11%) in the groups of feed addition; while acetylation was low in liver of the group of intramuscular injection.
814
OIKAWA, NAKAMOTO, HIROTA AND KATAGIRI
Doses were expressed as net value of SMX free base in the following text.
T-SMX - F-SMX = A-SMX where, T-SMX is total-SMX, F-SMX is freeSMX, and A-SMX is acetylated-SMX. Sensitivity limit for SMX by this method was 0. 1 p.p.m. in 5 grams wet specimens. Recovery rates were more than 82% in albumen and yolk, and 75% tissues, with 3 to 8% of coefficient of variation. Analytical procedure including the method for clean up was somewhat different for albumen and yolk, for fat and other tissues. The detailed procedure is reported by Sakano et al. (1976). RESULTS
1) Clearance in eggs. In the group fed 0.2%, the drug level in the albumen diminished abruptly to reach below 0.1 p.p.m. by day 3. On the other hand, the drug level in yolk remained almost at a plateau until day 4 and then diminished to below 0.1 p.p.m. by day 8.
c) Procedure for trials in tissues. SMX was administered to two groups of broiler chickens at a level of 0.2% and 0.4% in the feed for 5 successive days, respectively. At 10 a.m. of the final day of the medication, the drug was withdrawn and 5 chickens (3 males and 2 females) of each group were sacrificed at day 0. To another group of broiler chickens, SMX was given intramuscularly at a level of 100 mg. per kg. body weight, twice daily for 5 successive days. Two hours after the final administration was given, five chickens were sacrificed at day 0. Successively, five birds of each group were sacrificed at 1, 2, 3, 4 and 5 days after drug withdrawal. Tissue specimens of heart, liver, kidney, spleen, gizzard, fat, breast muscle and skin were stored in the refrigerator until analysis was done. 3. Analytic method. After the specimens of albumen, yolk and tissues were cleaned up, free-SMX in the specimens was treated with fluorescamine reagent, and fluorescent intensity was measured at 400—490 my., of excitation emission maxima by means of the Hitachi spectrofluorometer. After the cleaned up sample was treated with acid in a boiling water bath, total-SMX was measured by the same method as described above. Levels of the drug were determined by
0
1
2
3
4
5
6
7
8
9
10
Time in days after withdrawal of treatment
FIG. 1. Clearance curves of total-sulfamethoxazole in egg albumen and yolk of chicken after withdrawal of the drug given at 0.2% in feed for 5 successive days.
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b) Procedure for trials on eggs. SMX was administered to two groups of five hens each at a level of 0.2% (twice as much as the recommended dose) or 0.4% in the feed for 5 successive days, respectively (0.2% and 0.4% group). To another group of five chickens SMX was injected intramuscularly at a level of 100 mg. per kg. body weight, twice a day for 5 successive days (I.M. group). Eggs were collected daily at 10 a.m. throughout the experiment. At 10 a.m. of the final day of the medication, the drug added in the feed was withdrawn from the former two groups, and the final injection was given to the last group. The eggs collected by this time were named "eggs at day 0" of the drug withdrawal. Eggs were collected daily thereafter until day 10, and were stored in the refrigerator at 4—10° C. prior to analysis.
the formula:
SULFAMETHOXAZOLE CLEARANCE
815
The drug levels of both albumen and yolk in the group fed 0.4% were higher than those of the group fed 0.2% during the course of the drug withdrawal. It took longer time to reach 0.1 p.p.m. level in the higher dose than in the lower. In the group administered the drug I.M., the drug level in albumen decreased to below 0.1 p.p.m. by day 6. On the other hand, that in yolk remained at a plateau until day 2, and decreased gradually thereafter until day 4, and then dropped to below 0.1 p.p.m. by day 8-10. These clearance patterns of T-SMX in each bird are shown on the semilogarithmic scales in Figs. 1, 2 and 3. Average value for T- and F-SMX in albumen and yolk for corresponding days after drug withdrawal are listed in Tables 1 and 2, respectively. Proportion of A-SMX to T-SMX tended to increase with the lapse of time after drug withdrawal in both albumen and yolk without difference between medication routes or dose
Yolk Albumen
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FIG. 3. Clearance curves of total-sulfamethoxazole in egg albumen and yolk of chicken after withdrawal of the drug given at 100 mg./kg. intramuscularly, twice a day for 5 successive days.
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levels used (Fig. 4). Drug intake in the group fed 0.2% was estimated grossly 100 mg. per kg. per day because average food intake was of about 100 g. per day per bird weighing an average of 2 kg. Drug intake in the group fed 0.4% was almost equal to that in the group given I.M. at 100 mg. per kg. twice daily. 2) Clearance in tissue. T-SMX level in tissues at day 0 corresponded to the dose level. The drug level decreased abruptly, to below 0.1 p.p.m. by day 3 in heart, liver, gizzard, fat and muscle, and by day 4 in kidney, spleen and skin (Table 3). Proportion of A-SMX to T-SMX was rather high in liver, spleen, fat and skin in the groups fed 0.2% and 0.4%, whereas very low in kidney in the group of I.M. The large difference of acetylation in liver was observed between medication routes (Table 4).
Downloaded from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
AO
ABA
100
Mean ± standard deviation.
Number of eggs used.
Free-sulfamethoxazole (p.p.m.).
Total-sulfamethoxazole (p.p.m.).
Intramuscularly.
(5) 82.44 ± 28.45 80.07 ± 28.10
100 mg./kg./day X 2 i.m. a for 5 days T F (3) 47.69 ± 21.55 39.75 ± 18.45
(2) 22.47 ± 14.28 22.23 + 14.26
(5) 39.94 ± 11.09 38.95 + 10.84
0.4% in feed for 5 days T F
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(3) 11.91 ± 4.81 11.65 + 5.16
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(4) 2.72 + 2.44 2.55 ± 2.28
(4) 1.37 ± 0.90 1.31 ± 0.86
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(3) 0.26 0.23
(3) 0.10 <0.10
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Time in days after withdrawal of trea
in egg albumen of chicken after withdrawal of
0.2% in feed for 5 days
Treatment
TABLE 1. — Clearance of sulfamethoxazole
d from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
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TABLE 2. - Clearance of sulfamethoxazole
from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
0.17 + 0.12 0.17 + 0.10
11.82 ± 2.26 8.30 ± 1.60
12.66 ± 2.86 10.78 ± 2.30
1.99 ± 0.53 1.50+ 0.37
10.77 ± 2.93 9.74 ± 2.61
12.39 ± 3.34 9.01 ± 1.56
63.85 ± 30.96 56.87+31.16
T F
T F
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T F
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0.4% in feed Heart T F 0.22 + 0.10 0.18 ±0.11 0.72 + 0.36 0.61 + 0.29
41.13 + 23.46 23.57 ±14.58
132.67 ± 50.19 117.95 ±46.28
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T F
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0.12 + 0.03 0.10 ± 0.00
0.10 ±0.00 0.11 ±0.00
0.10 ± 0.01 0.11 ±0.01
0.12+0.03 0.11 ±0.01
0.62 ±0.23 0.54 ± 0.22
T F
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T F
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0.10 ± 0.00 0.10 ± 0.00
0.11 ±0.02 0.10 ±0.00
0.10 ± 0.00 0.10 ±0.00
0.10 ± 0.00 0.10 ±0.00
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TABLE 3. — Clearance of sulfamethoxazole
from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
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to 0.001% in feed), medication duration (from single medication to 30 days successive medication), analytical method and others. Residue or clearance of the sulfa drug depended upon experimental condition and kind of the drug so that the results appearing in the above papers were hard to compare and summarize. It may be concluded however, that although albumen and yolk were of the two main components in eggs, their clearance pattern of the drug differed due to the difference in their mode of formation and deposition in the metabolism of the drug by the ovary and oviduct, respectively. Clearance pattern of albumen was rather close to that of blood, and that of tissue was found to be a little delayed from them. Sulfa drugs are practically administered to chickens in their feed or water, orally, or intramuscularly. The recommended dose levels for coccidiosis in feed is 0.05% for sulfaquinoxaline and sulfadimethoxine and 0.1% for other sulfonamides, and that for leukocytozoon is 0.001%. A concentration of two to four times the recommended dose for coccidiosis is practically the highest dose that chicken can tolerate. Clearance studies designed in this work being administered for 5 successive days at 0.4% in feed may cover a practical use of the sulfa drug in poultry husbandry. Only a few reports have discussed the proportion of acetylated-sulfa drug in tissues and eggs of chickens. The proportion of acetylated sulfa drugs given chicken (1) differed among the kind of drugs (Onodera et al, 1970; Shaffer et al, 1950) (2) differed among medication routes in some tissues (the present work) (3) differed among tissues (Nakatani et al., 1963; Onodera et al, 1970; Shaffer et al, 1950; the present work) (4) tended to increase with time elapsed after drug withdrawal in both albumen and yolk (the present work). In the cases of sulfadimethoxine, sulfamonomethoxine, sulfamerazine (Onodera et al, 1970) and sulfamethoxazole (Nakatani et al, 1963), a higher proportion of acetylated- to total-sulfonamide was observed in the tissues of the digestive tract than in others. The Bratton-Marshall (B.M.) method (Bratton and Marshall, 1939) has been widely used for colorimetric determination of sulfa drugs in tissue, blood and eggs of chickens. To separate
SULFAMETHOXAZOLE CLEARANCE t h e sulfa drug from biological c o m p o n e n t s and from o t h e r drugs in t h e sample which react with t h e B.M. reagent, a clean u p p r o c e d u r e with a solvent system (Fellig and Westheimer, 1 9 6 8 ; Tishler et al, 1968) or thin layer c h r o m a t o g r a p h y (Phillips, 1975) were r e p o r t e d . By this m e t h o d , a large a m o u n t of specimens was n e e d e d t o d e t e c t t h e 0.1 p . p . m . sensitivity level.
ACKNOWLEDGEM ENT A u t h o r s wish t o t h a n k Mrs. H. Kawaguchi, M. Akahoshi, J. Ikeuchi, Y. Nishibe and Y. Matsuo for their technical assistance.
REFERENCES Blom. L., 1975. Plasma half-lives and the excretion into egg-white and -yolk of three sulphonamides and pyrimethamine after medication of laying hens. Acta Pharmacol. Toxicol. 3 7 : 7 9 - 9 3 . Bratton, A. C , and E. K. Marshall, Jr., 1939. A new coupling component for sulfonamide determination. J. Biol. Chem. 128:537-550. Fellig, J., and J. Westheimer, 1968. Determination of sulfadimethoxine in animal tissue. J. Agr. Food Chem. 16:738-740. Fellig, J., J. Westheimer, M. J. Walsh and W. L. Marusich, 1971a. Tissue clearance of Rofenaid in chicken and turkey. Poultry Sci. 50:1777—1783. Fellig, J., J. Westheimer, M. J. Walsh and R. A. Saperstein, 1971b. Determination of ormetoprim in a n i m a l t i s s u e . J . Agr. Food Chem. 19:1261-1263. Krieg, R., 1966. Sulfonamidgehalt in Eiern, Blut und Organen von Weissen Leghorn wahrend und nach einer Behandlung mit der Sulfonamid Sulmet (Sulfadimidin). 1. Mitteilung; Der Sulmetgehalt in dem Eiern von Weisen Leghorn. Arch. Geflugelk.
30:299-308. Krieg, R., and O. Siegmann, 1967. Der Sulfonamidgehalt im Blut, Eiern und Organen von Weissen Leghorn wahrend und nach einer Behandlung mit der Sulfonamid Sulmet (Sulfadimidin). 2. Mitteilung; Der Sulfmetgehalt im Blut und in den Geweben. Arch. Geflugelk. 31:166-172. Laurencot, H. J., A. Schlosser and J. L. lempstead, 1972. The deposition and clearance of Rofenaid in chicken and t u r k e y eggs. Poultry Sci. 51:1181-1189. Liider, H., K. W. Lai and K. H. Hinz, 1974. Blut und Gewebespiegel von Sulfamethezin und Sulfaquinoxaline bei Broilern nach Verbreichung der Medikamente uber das Trinkwasser. Zbl. Vet. Med. B, 21:101-118. Nakatani, Y., and J. Gotoh, 1963. Studies on the concentration in blood and the distribution in organs of sulfisomezole and sulfisomidine in chickens. J. Jap. Vet. Med. Assoc. 16:454—457. Oshima, Y., A. Kasahara, T. Onodera and M. Mogi, 1964. Experimental studies of sulfadimethoxine in fowls. I. Plasma concentration in hens after oral administration. Jap. J. Vet. Sci. 26:115—120. Onodera, T., S. Inoue, A. Kasahara and Y. Oshima, 1970. Experimental studies on sulfadimethoxine in fowls. III. Egg and other tissue levels of sulfadimethoxine in fowls. III. Egg and other tissue levels of sulfonamides. Jap. J. Vet. Sci. 32:275-283. Phillips, W. F., and J. E. Trafton, 1975. Drug residues in animal tissues. A screening method for sulfonamides extracted from animal tissues. J. Assoc. Offic. Anal. Chem. 58:44-47. Righter, H. F., J. M. Worthington and H. D. Mercer, 1971. Tissue-residue depletion of sulfamethazine in calves and chickens. Amer. J. Vet. Res. 32:1003-1006. Righter, H. F., J. M. Worthington, H. E. Zimmerman and H. D. Mercer, 1970. Tissue-residue depletion of sulfadimethoxine in poultry. Amer. J. Vet. Res. 31:1051-1054^ Sakano, T., S. Masuda, A. Yamaji, T. Amano, H. Oikawa and K. Nakamoto, 1976. Determination of residual Sulfisomezole (Sulfamethoxazole) in animal tissues with fluorescamine. Yakugaku Zassi, in press. Schlenker, F. A., and B. K. Simmons, 1950. The absorption, distribution and excretion of sulfaquinoxaline in poultry. Amer. J. Vet. Res. 11:291-295. Shaffer, J. M., and R. N. Bieter, 1950. Conversion of acetylsulfonamides to the unconjugated form by the chicken kidney. J. Pharm. Exp. Therap. 100:192-200. de Silva, J. A. F., and N. Strojny, 1975. Spectrofluorometric determination of pharmaceuticals containing aromatic or aliphatic primary amins group as their fluorescamine (Fluran) derivatives. Anal. Chem. 47:714-718. Tishler, F., J. L. Sutter, J. N. Bathish and H. E. Hagman, 1968. Improved method for determination of sulfonamides in milk and tissues. J. Agr. Food Chem. 1 6 : 5 0 - 5 3 . Yonezawa, S., H. Nakamura, H. Azechi and K. Ninomiya, 1968. Studies on blood levels of four sulfa drugs in chicks. Doyaku-ken Nempo. 5:154-160.
Downloaded from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
S o m e o t h e r m e t h o d s which did n o t use B.M. reagent were described. Fellig et al. ( 1 9 7 1 a,b) r e p o r t e d a s p e c t r o f l u o r o m e t r y of o r m e t h o p r i m by o x i d a t i o n with p e r m a n g a n a t e t o 4 , 5 dim e t h o x y - 0 - t o l u i c acid. De Silva ( 1 9 7 5 ) r e p o r t e d a f l u o r o m e t r y of various sulfa drugs using a fluorescamine reagent. Sakano et al. ( 1 9 7 6 ) described an improved m e t h o d for determining SMX in tissue and eggs using a fluorescamine reagent. Sensitivity limit of this m e t h o d is 0.1 p.p.m. in 5 g. w e t biological specimens. T h e present work utilized this m e t h o d . L a u r e n c o t ( 1 9 7 1 ) described a m e t h o d assayed s u l f a d i m e t h o x i n e labeled with radioactive carbon. Radioactivity was detected b y liquid scintillation c o u n t i n g t o a l o w of 0.01 p . p . m . A m o r e simplified a n d / o r sensitive m e t h o d for small a m o u n t s of biological m a t e r i a l has been developed for t h e residue s t u d y of sulfonamides in animal p r o d u c t s .
821
2
(Received for publication August 30, 1976)
Poultry Science 5 6 : 8 1 3 - 8 2 1 , 1977
INTRODUCTION Sulfamethoxazole (SMX) or sulfisomezole, a kind of sulfa drug, is a c h e m o t h e r a p e u t i c agent being used for t h e t r e a t m e n t of p r o t o z o a l and bacterial diseases of animals, and has been extensively used for t r e a t m e n t of chicken coccidiosis and infectious coryza. Considerable work on t h e clearance of t h e sulfa drug in t h e edible tissue a n d eggs of chickens was p e r f o r m e d (Blom, 1 9 7 5 ; Fellig et al., 1971 a,b; Krieg, 1 9 6 6 ; Krieg et al, 1 9 7 6 ; Laurencot et al., 1 9 7 2 ; Liider et al., 1 9 7 4 ; Nakatani et al., 1 9 6 3 ; Onodera et al, 1 9 7 0 ; a n d Righter et al, 1970, 1 9 7 1 ) . More detailed information is needed. T h e present s t u d y was u n d e r t a k e n t o determine t h e clearance patterns of SMX in egg a l b u m e n , y o l k and tissues after withdrawal of t h e drug in chicken t o which t h e drug was administered in t h e feed or intramuscularly for 5 successive days.
2.0 kg. in weight were used in t h e trial on eggs. Broiler chickens of b o t h sexes aged 4 0 t o 6 0 days old a n d 1.5 t o 2 . 0 kg. in weight were used in t h e trial o n tissues. These chickens purchased at a p o u l t r y farm in t h e n e i g h b o r h o o d were i n t r o d u c e d i n t o t h e single cage in t h e animal house of this laborat o r y t w o w e e k s before t h e m e d i c a t i o n . N o disease was observed. Animal h o u s e was maintained at 25±2 C. in r o o m t e m p e r a t u r e and 45—65% in h u m i d i t y . F o o d and water were taken ad lib. F o o d for layers was s u p p l e m e n t e d with o y s t e r shells a n d vitamins. 2. Medication and sampling, a) Drugs used. "Sinomin p o w d e r for animal u s e " containing SMX free base p r e m i x e d with starch p o w d e r at a 10% n e t c o n c e n t r a t i o n , and " S i n o m i n injection for animal u s e " containing SMX-monoe t h a n o l a m i n e a t a n e t value of 1 0 0 m g . per ml. as SMX free base were used. Chemical s t r u c t u r e of SMX is as follows.
MATERIALS AND METHODS 1. Animals and rearing conditions. White Leghorn h e n s , 7 t o 10 m o n t h s old a n d 1.5 t o
H2IM-// 7—S02 \=Z/
813
NH
n n N J^-CHa ^-0X
Downloaded from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
ABSTRACT Clearance of sulfamethoxazole (SMX, or sulfisomezole) in eggs and tissues of chicken after withdrawal of the drug which was medicated feed additively (at 0.2 or 0.4%) or administered intramuscularly (100 mg./kg., twice daily) for 5 successive days was determined using a fluorescamine reagent. Clearance patterns of SMX were found to be different between albumen and yolk. SMX level in albumen diminished linearly to below 0.1 p.p.m. (sensitivity level) by 5 days after the cessation of medication. On the other hand, SMX level in yolk remained at a plateau until 4 days, and thereafter decreased linearly to below 0.1 p.p.m. by 10 days. SMX levels in most tissues diminished below 0.1 p.p.m. by 3 days and those in kidney and skin by 4 days. Clearance patterns in tissue were quite similar to those in egg albumen. Proportion of acetylated-SMX to total-one in both albumen and yolk tended to increase up to 10% with the lapse of time after drug withdrawal. Acetylation was high in liver (36.5—43.6%), spleen skin and fat, but low in breast muscle and kidney (3.7—11%) in the groups of feed addition; while acetylation was low in liver of the group of intramuscular injection.
814
OIKAWA, NAKAMOTO, HIROTA AND KATAGIRI
Doses were expressed as net value of SMX free base in the following text.
T-SMX - F-SMX = A-SMX where, T-SMX is total-SMX, F-SMX is freeSMX, and A-SMX is acetylated-SMX. Sensitivity limit for SMX by this method was 0. 1 p.p.m. in 5 grams wet specimens. Recovery rates were more than 82% in albumen and yolk, and 75% tissues, with 3 to 8% of coefficient of variation. Analytical procedure including the method for clean up was somewhat different for albumen and yolk, for fat and other tissues. The detailed procedure is reported by Sakano et al. (1976). RESULTS
1) Clearance in eggs. In the group fed 0.2%, the drug level in the albumen diminished abruptly to reach below 0.1 p.p.m. by day 3. On the other hand, the drug level in yolk remained almost at a plateau until day 4 and then diminished to below 0.1 p.p.m. by day 8.
c) Procedure for trials in tissues. SMX was administered to two groups of broiler chickens at a level of 0.2% and 0.4% in the feed for 5 successive days, respectively. At 10 a.m. of the final day of the medication, the drug was withdrawn and 5 chickens (3 males and 2 females) of each group were sacrificed at day 0. To another group of broiler chickens, SMX was given intramuscularly at a level of 100 mg. per kg. body weight, twice daily for 5 successive days. Two hours after the final administration was given, five chickens were sacrificed at day 0. Successively, five birds of each group were sacrificed at 1, 2, 3, 4 and 5 days after drug withdrawal. Tissue specimens of heart, liver, kidney, spleen, gizzard, fat, breast muscle and skin were stored in the refrigerator until analysis was done. 3. Analytic method. After the specimens of albumen, yolk and tissues were cleaned up, free-SMX in the specimens was treated with fluorescamine reagent, and fluorescent intensity was measured at 400—490 my., of excitation emission maxima by means of the Hitachi spectrofluorometer. After the cleaned up sample was treated with acid in a boiling water bath, total-SMX was measured by the same method as described above. Levels of the drug were determined by
0
1
2
3
4
5
6
7
8
9
10
Time in days after withdrawal of treatment
FIG. 1. Clearance curves of total-sulfamethoxazole in egg albumen and yolk of chicken after withdrawal of the drug given at 0.2% in feed for 5 successive days.
Downloaded from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
b) Procedure for trials on eggs. SMX was administered to two groups of five hens each at a level of 0.2% (twice as much as the recommended dose) or 0.4% in the feed for 5 successive days, respectively (0.2% and 0.4% group). To another group of five chickens SMX was injected intramuscularly at a level of 100 mg. per kg. body weight, twice a day for 5 successive days (I.M. group). Eggs were collected daily at 10 a.m. throughout the experiment. At 10 a.m. of the final day of the medication, the drug added in the feed was withdrawn from the former two groups, and the final injection was given to the last group. The eggs collected by this time were named "eggs at day 0" of the drug withdrawal. Eggs were collected daily thereafter until day 10, and were stored in the refrigerator at 4—10° C. prior to analysis.
the formula:
SULFAMETHOXAZOLE CLEARANCE
815
The drug levels of both albumen and yolk in the group fed 0.4% were higher than those of the group fed 0.2% during the course of the drug withdrawal. It took longer time to reach 0.1 p.p.m. level in the higher dose than in the lower. In the group administered the drug I.M., the drug level in albumen decreased to below 0.1 p.p.m. by day 6. On the other hand, that in yolk remained at a plateau until day 2, and decreased gradually thereafter until day 4, and then dropped to below 0.1 p.p.m. by day 8-10. These clearance patterns of T-SMX in each bird are shown on the semilogarithmic scales in Figs. 1, 2 and 3. Average value for T- and F-SMX in albumen and yolk for corresponding days after drug withdrawal are listed in Tables 1 and 2, respectively. Proportion of A-SMX to T-SMX tended to increase with the lapse of time after drug withdrawal in both albumen and yolk without difference between medication routes or dose
Yolk Albumen
0
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FIG. 3. Clearance curves of total-sulfamethoxazole in egg albumen and yolk of chicken after withdrawal of the drug given at 100 mg./kg. intramuscularly, twice a day for 5 successive days.
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levels used (Fig. 4). Drug intake in the group fed 0.2% was estimated grossly 100 mg. per kg. per day because average food intake was of about 100 g. per day per bird weighing an average of 2 kg. Drug intake in the group fed 0.4% was almost equal to that in the group given I.M. at 100 mg. per kg. twice daily. 2) Clearance in tissue. T-SMX level in tissues at day 0 corresponded to the dose level. The drug level decreased abruptly, to below 0.1 p.p.m. by day 3 in heart, liver, gizzard, fat and muscle, and by day 4 in kidney, spleen and skin (Table 3). Proportion of A-SMX to T-SMX was rather high in liver, spleen, fat and skin in the groups fed 0.2% and 0.4%, whereas very low in kidney in the group of I.M. The large difference of acetylation in liver was observed between medication routes (Table 4).
Downloaded from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
AO
ABA
100
Mean ± standard deviation.
Number of eggs used.
Free-sulfamethoxazole (p.p.m.).
Total-sulfamethoxazole (p.p.m.).
Intramuscularly.
(5) 82.44 ± 28.45 80.07 ± 28.10
100 mg./kg./day X 2 i.m. a for 5 days T F (3) 47.69 ± 21.55 39.75 ± 18.45
(2) 22.47 ± 14.28 22.23 + 14.26
(5) 39.94 ± 11.09 38.95 + 10.84
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pc
(3) 11.91 ± 4.81 11.65 + 5.16
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(4) 2.72 + 2.44 2.55 ± 2.28
(4) 1.37 ± 0.90 1.31 ± 0.86
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(3) 0.26 0.23
(3) 0.10 <0.10
3
Time in days after withdrawal of trea
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0.2% in feed for 5 days
Treatment
TABLE 1. — Clearance of sulfamethoxazole
d from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
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TABLE 2. - Clearance of sulfamethoxazole
from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
0.17 + 0.12 0.17 + 0.10
11.82 ± 2.26 8.30 ± 1.60
12.66 ± 2.86 10.78 ± 2.30
1.99 ± 0.53 1.50+ 0.37
10.77 ± 2.93 9.74 ± 2.61
12.39 ± 3.34 9.01 ± 1.56
63.85 ± 30.96 56.87+31.16
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T F
T F
T F
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Fat
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41.13 + 23.46 23.57 ±14.58
132.67 ± 50.19 117.95 ±46.28
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0.10 ±0.00 0.11 ±0.00
0.10 ± 0.01 0.11 ±0.01
0.12+0.03 0.11 ±0.01
0.62 ±0.23 0.54 ± 0.22
T F
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T F
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6.51c 2.48 2.96 1.71
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0.10 ± 0.00 0.10 ± 0.00
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0.10 ± 0.00 0.10 ± 0.00
0.11 ±0.02 0.10 ±0.00
0.10 ± 0.00 0.10 ±0.00
0.10 ± 0.00 0.10 ±0.00
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Time in days after withdrawal of treatm
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Liver
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TABLE 3. — Clearance of sulfamethoxazole
from http://ps.oxfordjournals.org/ at University of British Columbia on July 3, 2015
819
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to 0.001% in feed), medication duration (from single medication to 30 days successive medication), analytical method and others. Residue or clearance of the sulfa drug depended upon experimental condition and kind of the drug so that the results appearing in the above papers were hard to compare and summarize. It may be concluded however, that although albumen and yolk were of the two main components in eggs, their clearance pattern of the drug differed due to the difference in their mode of formation and deposition in the metabolism of the drug by the ovary and oviduct, respectively. Clearance pattern of albumen was rather close to that of blood, and that of tissue was found to be a little delayed from them. Sulfa drugs are practically administered to chickens in their feed or water, orally, or intramuscularly. The recommended dose levels for coccidiosis in feed is 0.05% for sulfaquinoxaline and sulfadimethoxine and 0.1% for other sulfonamides, and that for leukocytozoon is 0.001%. A concentration of two to four times the recommended dose for coccidiosis is practically the highest dose that chicken can tolerate. Clearance studies designed in this work being administered for 5 successive days at 0.4% in feed may cover a practical use of the sulfa drug in poultry husbandry. Only a few reports have discussed the proportion of acetylated-sulfa drug in tissues and eggs of chickens. The proportion of acetylated sulfa drugs given chicken (1) differed among the kind of drugs (Onodera et al, 1970; Shaffer et al, 1950) (2) differed among medication routes in some tissues (the present work) (3) differed among tissues (Nakatani et al., 1963; Onodera et al, 1970; Shaffer et al, 1950; the present work) (4) tended to increase with time elapsed after drug withdrawal in both albumen and yolk (the present work). In the cases of sulfadimethoxine, sulfamonomethoxine, sulfamerazine (Onodera et al, 1970) and sulfamethoxazole (Nakatani et al, 1963), a higher proportion of acetylated- to total-sulfonamide was observed in the tissues of the digestive tract than in others. The Bratton-Marshall (B.M.) method (Bratton and Marshall, 1939) has been widely used for colorimetric determination of sulfa drugs in tissue, blood and eggs of chickens. To separate
SULFAMETHOXAZOLE CLEARANCE t h e sulfa drug from biological c o m p o n e n t s and from o t h e r drugs in t h e sample which react with t h e B.M. reagent, a clean u p p r o c e d u r e with a solvent system (Fellig and Westheimer, 1 9 6 8 ; Tishler et al, 1968) or thin layer c h r o m a t o g r a p h y (Phillips, 1975) were r e p o r t e d . By this m e t h o d , a large a m o u n t of specimens was n e e d e d t o d e t e c t t h e 0.1 p . p . m . sensitivity level.
ACKNOWLEDGEM ENT A u t h o r s wish t o t h a n k Mrs. H. Kawaguchi, M. Akahoshi, J. Ikeuchi, Y. Nishibe and Y. Matsuo for their technical assistance.
REFERENCES Blom. L., 1975. Plasma half-lives and the excretion into egg-white and -yolk of three sulphonamides and pyrimethamine after medication of laying hens. Acta Pharmacol. Toxicol. 3 7 : 7 9 - 9 3 . Bratton, A. C , and E. K. Marshall, Jr., 1939. A new coupling component for sulfonamide determination. J. Biol. Chem. 128:537-550. Fellig, J., and J. Westheimer, 1968. Determination of sulfadimethoxine in animal tissue. J. Agr. Food Chem. 16:738-740. Fellig, J., J. Westheimer, M. J. Walsh and W. L. Marusich, 1971a. Tissue clearance of Rofenaid in chicken and turkey. Poultry Sci. 50:1777—1783. Fellig, J., J. Westheimer, M. J. Walsh and R. A. Saperstein, 1971b. Determination of ormetoprim in a n i m a l t i s s u e . J . Agr. Food Chem. 19:1261-1263. Krieg, R., 1966. Sulfonamidgehalt in Eiern, Blut und Organen von Weissen Leghorn wahrend und nach einer Behandlung mit der Sulfonamid Sulmet (Sulfadimidin). 1. Mitteilung; Der Sulmetgehalt in dem Eiern von Weisen Leghorn. Arch. Geflugelk.
30:299-308. Krieg, R., and O. Siegmann, 1967. Der Sulfonamidgehalt im Blut, Eiern und Organen von Weissen Leghorn wahrend und nach einer Behandlung mit der Sulfonamid Sulmet (Sulfadimidin). 2. Mitteilung; Der Sulfmetgehalt im Blut und in den Geweben. Arch. Geflugelk. 31:166-172. Laurencot, H. J., A. Schlosser and J. L. lempstead, 1972. The deposition and clearance of Rofenaid in chicken and t u r k e y eggs. Poultry Sci. 51:1181-1189. Liider, H., K. W. Lai and K. H. Hinz, 1974. Blut und Gewebespiegel von Sulfamethezin und Sulfaquinoxaline bei Broilern nach Verbreichung der Medikamente uber das Trinkwasser. Zbl. Vet. Med. B, 21:101-118. Nakatani, Y., and J. Gotoh, 1963. Studies on the concentration in blood and the distribution in organs of sulfisomezole and sulfisomidine in chickens. J. Jap. Vet. Med. Assoc. 16:454—457. Oshima, Y., A. Kasahara, T. Onodera and M. Mogi, 1964. Experimental studies of sulfadimethoxine in fowls. I. Plasma concentration in hens after oral administration. Jap. J. Vet. Sci. 26:115—120. Onodera, T., S. Inoue, A. Kasahara and Y. Oshima, 1970. Experimental studies on sulfadimethoxine in fowls. III. Egg and other tissue levels of sulfadimethoxine in fowls. III. Egg and other tissue levels of sulfonamides. Jap. J. Vet. Sci. 32:275-283. Phillips, W. F., and J. E. Trafton, 1975. Drug residues in animal tissues. A screening method for sulfonamides extracted from animal tissues. J. Assoc. Offic. Anal. Chem. 58:44-47. Righter, H. F., J. M. Worthington and H. D. Mercer, 1971. Tissue-residue depletion of sulfamethazine in calves and chickens. Amer. J. Vet. Res. 32:1003-1006. Righter, H. F., J. M. Worthington, H. E. Zimmerman and H. D. Mercer, 1970. Tissue-residue depletion of sulfadimethoxine in poultry. Amer. J. Vet. Res. 31:1051-1054^ Sakano, T., S. Masuda, A. Yamaji, T. Amano, H. Oikawa and K. Nakamoto, 1976. Determination of residual Sulfisomezole (Sulfamethoxazole) in animal tissues with fluorescamine. Yakugaku Zassi, in press. Schlenker, F. A., and B. K. Simmons, 1950. The absorption, distribution and excretion of sulfaquinoxaline in poultry. Amer. J. Vet. Res. 11:291-295. Shaffer, J. M., and R. N. Bieter, 1950. Conversion of acetylsulfonamides to the unconjugated form by the chicken kidney. J. Pharm. Exp. Therap. 100:192-200. de Silva, J. A. F., and N. Strojny, 1975. Spectrofluorometric determination of pharmaceuticals containing aromatic or aliphatic primary amins group as their fluorescamine (Fluran) derivatives. Anal. Chem. 47:714-718. Tishler, F., J. L. Sutter, J. N. Bathish and H. E. Hagman, 1968. Improved method for determination of sulfonamides in milk and tissues. J. Agr. Food Chem. 1 6 : 5 0 - 5 3 . Yonezawa, S., H. Nakamura, H. Azechi and K. Ninomiya, 1968. Studies on blood levels of four sulfa drugs in chicks. Doyaku-ken Nempo. 5:154-160.
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S o m e o t h e r m e t h o d s which did n o t use B.M. reagent were described. Fellig et al. ( 1 9 7 1 a,b) r e p o r t e d a s p e c t r o f l u o r o m e t r y of o r m e t h o p r i m by o x i d a t i o n with p e r m a n g a n a t e t o 4 , 5 dim e t h o x y - 0 - t o l u i c acid. De Silva ( 1 9 7 5 ) r e p o r t e d a f l u o r o m e t r y of various sulfa drugs using a fluorescamine reagent. Sakano et al. ( 1 9 7 6 ) described an improved m e t h o d for determining SMX in tissue and eggs using a fluorescamine reagent. Sensitivity limit of this m e t h o d is 0.1 p.p.m. in 5 g. w e t biological specimens. T h e present work utilized this m e t h o d . L a u r e n c o t ( 1 9 7 1 ) described a m e t h o d assayed s u l f a d i m e t h o x i n e labeled with radioactive carbon. Radioactivity was detected b y liquid scintillation c o u n t i n g t o a l o w of 0.01 p . p . m . A m o r e simplified a n d / o r sensitive m e t h o d for small a m o u n t s of biological m a t e r i a l has been developed for t h e residue s t u d y of sulfonamides in animal p r o d u c t s .
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