J. Anim. Sci. 2002. 80:2931-2941
© 2002 American Society of Animal Science
Binding of ractopamine HCl to ocular tissues of cattle and turkeys in vivo and to melanin in vitro1,2
D. J. Smith3,
K. M. Ehrenfried,
J. D. Dalidowicz4 and
M. P. Turberg5
Elanco Animal Health, Greenfield, Indiana 46140
5 Correspondence:
2001 West Main Street (phone: 317-277-5041; fax: 317-277-4993; E-mail:
mturberg{at}lilly.com).
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Abstract
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Experiments were conducted to determine the total residues remaining in ocular tissues of cattle and turkeys after oral administration of [14C]ractopamine HCl. Twelve cattle were intraruminally dosed with 0.9 mg•kg-1•d-1 of [14C]ractopamine HCl for 7 d. Four cattle each were slaughtered with withdrawal periods of 48, 96, and 144 h. Radioactive residues were not detectable in whole-eye homogenates from the cattle. Eight male and eight female turkeys per treatment received either 7.5, 22.5, or 30 ppm dietary [14C]ractopamine HCl (0.33, 1.02, and 1.36 mg•kg-1•d-1; treatment groups 1, 2, and 3, respectively) for 7 d, and the birds were slaughtered with a 0-d withdrawal period. Eyes were dissected into retina/choroid/schlera (RCS), cornea/iris (CI), and aqueous humor (AH) fractions. Residues in RCS, CI, and AH of treatment 1 turkeys were not detectable. Residues in AH were < 0.02 ppm in treatment groups 2 and 3. Mean residues in RCS ranged from 0.15 to 0.26 ppm, and mean CI residues ranged from <0.09 to 0.17 ppm for treatment groups 2 and 3, respectively. The propensities of ractopamine and synthetic ractopamine metabolites to bind to melanin were studied in vitro using radiolabeled ligands with centrifugal filtration to separate melanin from unbound ligand. In vitro studies showed that [14C]ractopamine HCl binds to melanin rapidly and was displaced from melanin by other ß-agonists. Glucuronidation of ractopamine, which produced the major biotransformation product of ractopamine in all species studied to date, prevented binding to melanin. These studies demonstrate that the propensity for the in vivo binding of ractopamine HCl to pigmented ocular tissues is less than that reported for clenbuterol.
Key Words: Cattle • Clenbuterol • Melanins • Ractopamine • Residues • Turkeys
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Introduction
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Several studies have shown that ß-adrenergic agonists such as clenbuterol, salbutamol, and terbutaline may accumulate in pigmented ocular tissues of treated animals (Meyer and Rinke, 1991; Malucelli et al., 1994). Depletion of clenbuterol from ocular tissues of farm animals occurs at much slower rates than its depletion from edible tissues, such as liver or kidney. Indeed, clenbuterol residues in eyes of treated animals are measurable weeks after residues in liver and kidney have declined to undetectable levels (Elliott et al., 1993a,b; Malucelli et al., 1994). For this reason, monitoring ocular tissues from animal carcasses has been very useful for detecting the illegal use of clenbuterol in Europe and the United States (Kuiper et al., 1998; Mitchell and Dunnavan, 1998).
Available data suggest that not all ß-adrenergic agonists have the same propensity to accumulate in ocular tissues (Howells et al., 1994; Sauer and Anderson, 1994; Polettini et al., 1995). Therefore, it was of interest to determine whether residues of the phenethanolamine ß-agonist ractopamine HCl (Figure 1
) would also accumulate in the eyes of test animals during experimental exposure. To this end, the total radioactive residues of ractopamine HCl were measured in the eyes of cattle and turkeys after experimental exposure to dietary [14C]ractopamine HCl. In order to account for the apparent low propensity of ractopamine to bind to ocular tissues, relative to the reported binding of clenbuterol, the in vitro binding of [14C]ractopamine HCl and [14C]ractopamine glucuronide to and displacement from isolated melanin was studied. Melanin was used in in vitro studies because it has been identified as a critical factor in the in vivo binding of drugs to pigmented matrices such as hair and pigmented tissues of the eye (Ings, 1984).
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Figure 1. Chemical structures of ractopamine HCl (top) and other ß-agonists used in the in vitro portion of this study. Common to all phenethanolamine ß-agonists are a substituted phenyl ring, a benzylic alcohol, and a secondary amine. The substituted phenyl group and benzylic hydroxyl group are mandatory for ß-agonist activity and are on the ß-carbon relative to the secondary amine.
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Materials and Methods
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Cattle Study
Animal Handling.
Sixteen Hereford heifers (n = 8) and steers (n = 8) were each fitted with ruminal cannula. After a 7-wk recovery period, six animals of each sex (212.3 ± 18.6 kg) were acclimated to individual metabolism crates for 9 d. One steer, a replacement for a steer whose ruminal cannula started to leak, was acclimated to its metabolism crate for a 3-d period. During the acclimation periods, all cattle were provided ad libitum access to water and the ration shown in Table 1. Control animals were housed and managed similarly to treated animals with the exception of receiving no dietary ractopamine. Prior to the start of the study the research protocol was approved by the Institutional Animal Use Committee.
Dose and Dose Administration.
Radioequivalent quantities of two [14C]ractopamine HCl lots, each lot radiolabeled in a separate phenyl ring, were dissolved in ethanol and were diluted with nonradioactive ractopamine to a specific activity of 0.25 µCi/mg (0.6 dpm/ng). The radiochemical purity of each [14C]ractopamine HCl was greater than 98%. Aliquots of the radiolabeled ractopamine solution equivalent to one-half of the daily dose for each animal were placed into gelatin capsules containing feed, and the ethanol was allowed to dry. Animals were dosed each morning and evening by placing a gelatin capsule containing one-half of the daily [14C]ractopamine HCl dose into the rumen via the ruminal cannula. Intraruminal dosing was chosen to avoid problems associated with repeated gavage dosing using a balling gun. Each animal was dosed twice daily for seven consecutive days with a total daily dose equivalent to 0.9 mg/kg body weight. Animals were provided basal feed during the treatment and withdrawal periods.
Slaughter and Tissue Collection.
Two steers and two heifers were each slaughtered 48, 96, and 144 h after the last administration of [14C]ractopamine. A control heifer and a control steer were slaughtered the same day as the animals with the 48-h withdrawal period. Animals were slaughtered by captive bolt stunning followed by exsanguination and edible tissues were collected for residue analysis; the eyes from each animal were also collected. All tissues were frozen at -20°C until analysis.
Radiochemical Analysis of Eyes.
Whole eyes were trimmed of connective tissue and were homogenized in a Waring Commercial Blender (Waring; New Hartford, CT). Cattle eyes were not dissected because at the time of the study it was believed that residue-monitoring programs would most likely utilize whole-eye homogenates. Triplicate subsamples (0.5 g) of the eye homogenates were solubilized in 3 mL of Soluene 350 Tissue solubilizer (Packard Instrument Co., Downers Grove, IL) over several days. The solubilized tissues were diluted with 15 mL of a scintillator solution composed of 600 mg 2,5-diphenyloxazole (Fisher Scientific; Fair Lawn, NJ) and 7.5 mg of 1,4-bis-2(4-methyl-5-phenyloxazolyl)benzene (Mallinckrodt; Paris, KY) per 100 mL of toluene. Radioactivity in the samples was quantified on a Packard Tri-Carb Scintillation Counter (Packard Instrument Co., Downers Grove, IL). Quench was corrected by the addition of an internal standard.
Background radioactivity was determined by counting five replicate 0.5-g samples of eye homogenate from control animals, processed as described above. The limit of detection was defined as the mean background counts per minute (cpm) of control eyes plus three standard deviations or plus 4 cpm, whichever was greater. Mean sample counts per minute from test animals that were less than the limit of detection were considered to have no detectable residues.
Total radioactive residues in cattle liver and kidney were measured by combustion analysis of 0.25- to 0.5-g homogenized tissue samples. Determination of radioactivity was performed as described above.
Turkey Study
Animal Handling and Housing.
Toms (approximately 11.0 kg and 16 wk of age) and hens (approximately 7.0 kg and 14 wk of age) were obtained from Perdue Farms, Washington, Indiana. Upon delivery to the test site, hens and toms were individually identified with numbered wing tags, weighed, and were randomly allotted within sex to concrete floor pens. Each pen was bedded with pine shavings and was equipped with a galvanized steel gravity feeder and a Plasson automatic watering system. The birds were allowed ad libitum access to a basal ration (Table 2) and water during the acclimation period. Pen feed consumption was measured during the acclimation and treatment periods.
[14C]Ractopamine HCl Preparation.
Radiochemically equivalent quantities of two lots of [14C]ractopamine HCl (radiochemical purities greater than 95%), with each lot containing the radiocarbon in a separate phenyl ring, were mixed with nonradiolabeled ractopamine HCl to a specific activity of 0.30 µCi/mg (0.67 dpm/ng). A [14C]ractopamine HCl stock solution was formed by diluting the mixture with ethanol to volume in a 500-mL volumetric flask.
Feed Preparation.
Three batches of basal feed (Table 2
) were prepared to contain 7.5, 22.5, and 30 ppm of [14C]ractopamine. Individual batches of feed were prepared by transferring an appropriate volume of the [14C]ractopamine HCl stock solution to a 250-mL volumetric flask and diluting to the mark with ethanol. The volume of stock solution transferred contained the appropriate mass of [14C]ractopamine HCl to formulate diets containing either 7.5, 22.5, or 30 ppm of ractopamine. The correct mass of basal turkey feed was weighed, added to a ribbon mixer, and a portion (approximately 50 mL) of the appropriate [14C]ractopamine dilution was added to the surface of the feed. Ethanol was allowed to evaporate, and the feed was mixed for approximately 20 min. This process was repeated until all of the [14C]ractopamine dilution was added to the feed. The volumetric flask containing the ractopamine dilution was rinsed with 50 to 75 mL of ethanol, and the rinse was added to the feed, allowed to dry, and mixed as described. Feed batches were mixed from the lowest ractopamine concentration to the highest ractopamine concentration on three consecutive workdays.
Dosing.
Prior to the initiation of treatments, all birds were reweighed. Animals were segregated by sex into six pens and treatment groups were randomly allocated to pens so that two pens received each treatment. Within pens, eight birds were randomly selected to participate in the study, and six birds of each sex were randomly selected for inclusion in control groups; extra birds were removed from each pen and were euthanized by CO2 induced asphyxiation. Test animals in treatments 00, 01, 02, and 03 were given ad libitum access to feed containing 0.0, 7.5, 22.5, and 30.0 ppm of [14C]ractopamine HCl, respectively, for seven consecutive days. Dietary ractopamine concentrations were chosen to bracket those described by Wellenreiter and Tonkinson (1990). Partitions present between pens prevented contact among animals of different treatments and also prevented radiochemical cross contamination of pens. Feed intakes (2.59 ± 0.22 and 3.78 ± 0.32 kg for hens and toms, respectively) and animal weights at the beginning and the end of the study were recorded (Table 3). Prior to the start of the study, a protocol was approved by the Institutional Animal Use Committee.
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Table 3. Mean (±SD) starting and ending weights of male and female turkeys provided 7.5, 22.5, and 30 ppm [14C]dietary ractopamine HCl for seven consecutive days
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Slaughter.
At the end of the 7-d treatment period, feed was withdrawn from each pen of birds approximately 6 h prior to slaughter. To maintain a 6-h withdrawal period, feed removal was staggered so that the order of slaughter was controls followed by treatments 01, 02, and 03, respectively. Prior to slaughter by electrical stunning and exsanguination, the weight of each bird was measured and recorded. Edible tissues and eyes were collected and frozen at -20°C until analysis. This report will address results obtained from the analysis of ocular tissues of ractopamine treated turkeys, but will not report residues present in edible tissues.
Analysis of Eyes.
Control eyes were processed from two randomly selected control males and females. Eyes from control and treated birds were thawed, dissected free of extraneous connective tissue, and both eyes from each bird were dissected into aqueous humor (AH), cornea/iris (CI), lens, and retina/choroid/schlera (RCS) fractions.
The AH was removed from eyes using disposable syringes (3 mL) fitted with a 21-gauge needle. The needle was inserted into the vitreous body of the eye, and the aqueous humor was drawn into the syringe. The AH, was displaced from each syringe into tared scintillation vials so that three, approximately 1-g samples of AH were collected per eye. The mass of each AH aliquot was recorded. Samples were dissolved in 3 mL of Soluene 350 (Packard; Downer’s Grove, IL) and were allowed to digest for at least 1 wk. Solubilized AH samples were diluted in 15 mL of a toluene-based liquid scintillation fluid, placed in the dark, and chilled several hours prior to liquid scintillation counting. Each sample was counted for 10 min by liquid scintillation counting (Packard Model 1900; Packard; Downer’s Grove, IL), and quench was corrected by the addition of an internal standard to each sample.
The CI and lens fractions were dissected from the eyes, placed into tared scintillation vials, weighed, and frozen until analysis. Radioactivity was measured by solubilization in 3 mL of Soluene 350 followed by dilution in 15 mL of a toluene-based liquid scintillation fluid of the composition described for analysis of cattle eye homogenates. Samples were counted and quench was corrected as described above for the aqueous humor. Lens samples contained sufficient quantities of quench, even after the addition of internal standard, that the reliable quantitation of residues was not possible.
The RCS fraction of each eye was cut into three pieces; each piece was placed into a tared scintillation vial and weighed, and the samples were solubilized as described above. Solubilized RCS was extremely dark indicating that reliable liquid scintillation counting would be impossible due to quench. Therefore a preliminary experiment was conducted in which RCS samples of control birds were solubilized and duplicate aliquots of the solubilized tissue, ranging from 125 to 1000 µl, were each fortified with varied amounts of [14C]ractopamine (approximately 70, 140, 280, 560, 110, and 2200 dpm). The fortified solubilized tissues were then diluted in 15 mL of the toluene-based liquid scintillation fluid described above, and the samples were counted. Quench was corrected using an internal standard. Results from the experiment (data not shown) indicated that 1000-µl samples of solubilized RCS could be counted with an acceptable recovery of radioactivity the lowest fortified level (110%; the range of recoveries for the various fortification levels was 101 to 112% of theoretical). For the analysis of the RCS, 1000-µl aliquots were removed from the solubilized tissues and diluted in 15 mL of a toluene-based liquid scintillation fluid. Quench was corrected with the use of an internal standard.
The objectives of this study were to determine whether ractopamine residues were present ocular tissues after ractopamine feeding and statistical differences between treatment means were not determined. If treatment differences were present, the interpretation and use of the data would not be changed.
Ractopamine Binding to Melanin Particles
Time Course for Ractopamine Binding.
Binding of [14C]ractopamine HCl to melanin from Sepia officinalis (Sigma Chemical Co., St. Louis, MO) was determined by the following procedure. Aliquots (2 mL) of a 5.0 x 10-6 M [14C]ractopamine solution, dissolved in 0.1 M potassium phosphate buffer, pH 7.4, were combined with 2-mL aliquots of buffer in 16 x 100 mm polypropylene tubes. A 1-mL aliquot of a 0.4 mg/mL melanin suspension (in buffer) was added to each tube. Triplicates were prepared, vortexed, and allowed to sit at room temperature for either 0, 10, 20, 30, 45, 60, 120, 180, 240, 300, or 360 min. At the termination of each incubation, the contents of each tube were transferred to the sample chamber of a 5-mL centrifugal filter tube (0.2 or 0.45 µm; Rainin; Woburn, MA) and the tubes were centrifuged for 5 min at approximately 200 x g (International Equipment Company; Needham Heights, MA). One-milliliter aliquots of the filtrates were removed from the receiving tubes of the filter units and were diluted in 15 mL of liquid scintillation fluid (Ready Solve, Beckman Instruments; Fullerton, CA). Radioactivity in each sample was determined by liquid scintillation counting, and quench was corrected with the use of an internal standard. [14C]Ractopamine and [14C]ractopamine glucuronide binding to the filters was determined in the absence of melanin and was less than 2.3% of the total radiocarbon loaded.
Competition Assays.
All reagents for competition assays were prepared in 0.1 M potassium phosphate buffer, pH 7.4. Duplicate 2-mL aliquots of a 5 x 10-7 M [14C]ractopamine solution were combined with 2-mL aliquots of 5.0 x 10-3, 5.0 x 10-4, 5.0 x 10-5, 5.0 x 10-6, 5.0 x 10-7, 5.0 x 10-8, and 5.0 x 10-9 M solutions of either clenbuterol HCl, fenoterol HBr, ritodrine HCl (Sigma Chemical Co., St. Louis, MO), isoproterenol HCl (Aldrich Chemical Co., Milwaukee, WI), or cimaterol (Tocris Cookson Inc., St. Louis, MO). One milliliter of a 0.4 mg/mL suspension of melanin (from Sepia officinalis; Sigma Chemical Company; St. Louis, MO) was added to each tube, and the tubes were vortexed. For each set of competition assays, duplicate control tubes with buffer substituted for the nonradioactive ß-agonists (no competition) were run. At the end of a 45-min incubation period, the contents of each tube were transferred to a centrifugal filter tube and radioactivity in the samples was processed as described above.
Binding of Ractopamine Glucuronides to Melanin.
[14C]Ractopamine-glucuronides (501 dpm/µg of ractopamine HCl equivalents) were synthesized and characterized in detail as described by Smith et al. (1993) except that swine liver was used in place of rabbit liver. The synthetic glucuronides consisted of a mixture of glucuronides conjugated to either phenolic ring of ractopamine, but no diconjugates and no conjugates to the ß-hydroxyl group were present. Radiochemical purity of the glucuronides was greater than 95% as assessed by reversed phase HPLC (95% 0.05 M ammonium acetate, pH 4.5 and 5% acetonitrile, ramped to 50% acetonitrile over 25 min using a linear gradient; 3.9 mm x 30 cm Waters Nova-Pak C-18 column; flow rate 1.0 mL/min) and liquid scintillation counting of fractions trapped off the column.
Stock solutions of ractopamine-glucuronides containing approximately 333 and 3300 ng per 2 mL of 0.1 M potassium phosphate buffer, pH 7.4, were made up in volumetric flasks, and the concentrations of radioactivity were validated by liquid scintillation counting. Two-milliliter aliquots of each [14C]ractopamine-glucuronide solution were combined and vortexed, with 2 mL of 0.1 M potassium phosphate buffer, pH 7.4. Aliquots (1.0 mL) of a 0.4 mg/mL melanin suspension were then added the tubes, and the tubes were vortexed. Control tubes contained buffer in place of the melanin suspension so that nonspecific binding could be determined. After a 45-min incubation period the contents of each tube were transferred to a 0.2-µm centrifugal filtration tube (Rainin; Woburn, MA), and the melanin was separated by filtration at approximately 200 x g. Aliquots (2 mL) of the filtrate were assayed to determine the amount of free [14C]ractopamine-glucuronide remaining in solution.
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Results
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Cattle Study.
Mean concentrations of total radioactive residues in whole-eye homogenates of cattle slaughtered 48, 96, and 144 h after withdrawal from [14C]ractopamine HCl are shown in Table 4 with the corresponding limit of detection. No radioactive residues were detected in whole-eye homogenates of any of the cattle used in this experiment.
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Table 4. Limits of detection, mean gross cpm, and total radioactive residues present in ocular homogenates of cattle dosed intraruminally with 0.9 mg•kg-1•d-1 of [14C]ractopamine HCl for seven consecutive days and slaughtered with withdrawal periods of 48, 96, and 144 h
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Turkey Study.
Mean concentrations of total radioactive residues in ocular tissues of male and female turkeys are shown in Table 5. Values for lenses are not reported because extensive sample quenching prevented reliable measurement of the radioactive residues in lenses. No radioactive residues were detected in any of the ocular tissues of turkeys fed 7.5 ppm [14C]ractopamine HCl for seven consecutive days. Mean concentrations of residues in AH were below 0.02 ppm in turkeys treated with 22.5 and 30 ppm of [14C]ractopamine HCl. Residues in RCS tissues averaged 0.17 and 0.25 ppm in the 22.5 and 30 ppm treatment groups, respectively. Residues in the CI of turkeys averaged 0.11 ppm in the 22.5 ppm treatment group and averaged 0.17 ppm in the 30 ppm treatment group.
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Table 5. Concentrations (ppm) of total radioactive residues (ractopamine HCl equivalents; mean ± SD) in ocular tissues of turkeys after exposure to 7.5, 22.5, or 30 ppm [14C] dietary ractopamine HCl for seven consecutive days (8 male and female turkeys per treatment)
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In Vitro Binding Assays
The time course for ractopamine HCl binding to melanin is shown in Figure 2. [14C]Ractopamine binding was determined by measuring free ractopamine in solution after removal of melanin from the suspension by centrifugal filtration. Because the amount of ractopamine added to each tube was known, the amount bound was readily calculated. Maximal binding to melanin was achieved within 10 min, and there was no major difference in binding from 10 min to 6 h. At time 0 (T0), 39% of the radioactivity was bound to melanin, indicating that the association between the melanin and ractopamine was rapid. The T0 measurement was made by combining all of the reagents directly in a centrifugal filtration tube already in place in the centrifuge. Immediately upon the addition of the melanin, the door to the centrifuge door was closed, and the unit was started. Thus, the association between the [14C]ractopamine HCl and the melanin was very rapid.
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Figure 2. Time course for ractopamine HCl binding to melanin from Sepia officinalis. Association between ractopamine and melanin, under the conditions described in the material and methods, was rapid and reached plateau values within 10 min. At T0 nearly 40% of the ractopamine was bound.
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The displacement of [14C]ractopamine HCl from melanin by clenbuterol HCl, cimaterol, isoproterenol HCl, fenoterol HBr, and ritodrine HCl (Figure 1) is shown in Figure 3. Ritodrine HCl and fenoterol HBr displaced [14C]ractopamine more readily than isoproterenol HCl, cimaterol, or clenbuterol HCl. It is noteworthy that fenoterol and ritodrine are both substituted with two substituted phenyl rings, much like ractopamine. Isoproterenol, clenbuterol, and cimaterol, however, are both substituted with only one phenyl ring.
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Figure 3. Displacement of [14C]ractopamine HCl from melanin from Sepia officinalis by clenbuterol, cimaterol, isoproterenol, fenoterol, and ritodrine (structures shown in Figure 1). Displacement of ractopamine HCl from melanin by ß-adrenergic agonists with two phenyl groups was more efficient than displacement by ß-agonists with only one phenyl group.
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The relative affinities of [14C]ractopamine HCl and synthetic [14C]ractopamine-glucuronides are shown in Figure 4. When 333 ng or 3,468 ng of [14C]ractopamine HCl was incubated with 0.4 mg of melanin, 71 and 61% of the radioactivity, respectively, remained associated with the melanin particles. In contrast, when 333 or 3,320 ng of [14C]ractopamine-glucuronide was combined with 0.4 mg of melanin, only 0 and 7.2% of the radioactivity remained bound to the melanin, respectively. Thus, the affinity of ractopamine HCl and its major metabolite, ractopamine glucuronide, for melanin differed substantially. Nonspecific binding in incubations containing no melanin was less than 2% of the total radioactvitiy added.
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Figure 4. Relative binding of [14C]ractopamine HCl and [14C] ractopamine glucuronides to melanin. Equimolar concentrations of ractopamine HCl and ractopamine glucuronides (calculated as ractopamine HCl equivalents) were incubated with fixed amounts of melanin. When 333 ng of ractopamine was incubated with melanin, over 70% of the radioactivity remained associated with the filtered melanin, but when the same mass of ractopamine glucuronide (calculated as ractopamine HCl equivalents) was incubated with melanin, 0% of the radioactivity was associated with the filtered melanin. Greater than 60% of the radioactivity was associated with melanin when the mass of ractopamine HCl was increased to 3,468 ng per incubation. When ractopamine glucuronides (3,320 ng) were allowed to associate with melanin, about 7% of the radioactivity was bound to melanin.
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Discussion
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Ractopamine Binding to Ocular Tissues
Residues in Cattle.
Previous publications have provided convincing evidence that the phenethanolamine ß-agonist clenbuterol accumulates in and depletes rather slowly from the eyes of cattle (Elliott et al., 1993c; Meyer and Rinke, 1991; Sauer and Anderson, 1994), chickens (Malucelli et al., 1994), and swine (Smith, 2000). Because clenbuterol depletes from eyes at a significantly lower rate than it depletes from urine, liver, and kidneys, ocular clenbuterol residues remain at measurable concentrations for several weeks. Therefore, the eye has been used as a tissue for monitoring the illegal use of clenbuterol (Kuiper et al., 1998; Mitchell and Dunnavan, 1998). Elliott et al. (1993a) demonstrated the utility of the eye for monitoring clenbuterol use by showing that clenbuterol residues in the retina of cattle ranged from 55 to 123 ppb after a 56-day withdrawal period; clenbuterol residues in livers of the same animals were only 0.3 to 0.4 ppb.
Our data clearly indicate that radioactive residues were not detectable in whole-eye homogenates of cattle fed [14C]ractopamine HCl for seven consecutive days (Table 4). A 7-d period was chosen as an appropriate length of time for dosing because liver residues are at a steady state after a 7-d dietary exposure to ractopamine HCl (Dalidowicz and Thomson, unpublished observations). Radioactive residues were measurable in liver and kidney homogenates at each withdrawal period; thus, residues of ractopamine were, by definition, greater in these tissues.
Given the fairly low specific activity [14C]ractopamine used in the cattle study, the limit of quantitation of ractopamine equivalents in eye homogenates from cattle was approximately 20 ppb (Table 4). Recent work by Churchwell et al. (2002) indicates that cattle fed 20 ppm ractopamine do accumulate some ractopamine residue in retinal tissue. Churchwell et al. (2002) measured retinal ractopamine in cattle fed 20 ppm dietary ractopamine for seven consecutive days. Retinal ractopamine concentrations were between 0.5 and 1 ppb in animals slaughtered after withdrawal periods of 0, 3, or 7 d. Thus, ocular tissues may be used to monitor cattle exposure to ractopamine, but sensitive analytical methods such as liquid chromatography mass spectrometry or immunoassay will be required.
In clenbuterol-treated calves, parent clenbuterol concentrations in whole-eye homogenates exceeded clenbuterol concentrations in liver by factors of 3.1 and 35.4 at 0 and 3.5 d of withdrawal, respectively (Meyer and Rinke, 1991). Data in Table 6 summarizes a number of clenbuterol residue studies providing additional evidence that clenbuterol accumulates preferentially in eyes of cattle. Regardless of the dose or duration of clenbuterol administration, or the length of the withdrawal period after termination of clenbuterol treatment, clenbuterol residues were consistently greater in eyes of cattle than in liver or kidney tissues. In contrast, ractopamine residues in ocular tissues were low relative to clenbuterol concentrations in ocular tissues. Ocular ractopamine residues were also low relative to concentrations of liver and kidney residues in animals slaughtered with no withdrawal period. Differences in the ocular, hepatic, and renal accumulation of the two phenethanolamine ß-agonists are accentuated by the fact that ractopamine treated cattle in this study received oral doses of 900 µg/kg BW per day. This dose is approximately 45 times the maximum level of clenbuterol administered to cattle in the cited studies (Table 6). In addition, only parent clenbuterol was measured in the cited studies, but the total radioactive residue of ractopamine was measured in the edible tissues and eyes of ractopamine treated cattle. Similarly, in the study of Churchwell et al. (2002), cattle were treated with 390 µg/kg body weight of ractopamine per day with subsequent retinal residues of parent ractopamine of about 1 ppb, regardless of the withdrawal period.
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Table 6. Comparison of residues of clenbuterol in tissues of clenbuterol-treated cattle with the total residues present in tissues of ractopamine HCl-treated cattle
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Clenbuterol accumulates specifically in the retina/choroid. The aqueous and vitreous humor do not sequester clenbuterol to any appreciable degree (Sauer and Limer, 1993; Biolatti et al., 1994). Thus, measurement of residues in whole-eye homogenates, as conducted in the present study, could substantially dilute any residue concentrated in the choroid/retina. Such a dilution might also explain the absence of ractopamine residues observed in the ocular homogenates of cattle used in this study. Data compiled in Table 6 were mostly from studies in which clenbuterol residues were specifically measured in the choroid/retina. Clenbuterol residues in these tissues are quite concentrated, sometimes exceeding 1 µg/g (1 ppm). However, in studies where clenbuterol was measured in homogenates of the whole eye (Meyer and Rinke, 1991), residues ranged from 118 to 15 ppb at 0- and 14-d withdrawal periods, respectively. In whole-eye homogenates of broilers, clenbuterol, salbutamol, and terbutaline residues were 90, 85, and 22 ppb, respectively, (Malucelli et al., 1994) at 0-d withdrawal periods. Thus, if ractopamine accumulated in cattle eyes in proportion to clenbuterol, it is reasonable to expect that it should have been detected in the current study.
Residues in Turkeys.
In contrast to residues in the eyes of cattle, radioactive residues were present in ocular tissues of turkeys. Residues in turkey ocular tissues followed a dose-response pattern with residues in the 7.5, 22.5, and 30.0 ppm dietary ractopamine treatments having no detectable residues, marginally detectable residues, and consistently detectable residues, respectively, in the CI tissues. Residues in the RCS were the most concentrated compared to other tissues.
The oral doses of ractopamine HCl received by the birds in the current study, expressed on a mg/kg BW (per day) basis, were 0.328, 1.02, and 1.36, for the 7.5, 22.5, and 30 ppm treatment groups, respectively. These doses bracket the dose of ractopamine HCl given to cattle in this study (0.9 mg/kg) and, in the case of the highest dose, is from 85 to 272 times the daily level of clenbuterol dosed in the studies cited in Table 6. Compared to the ocular residues present in ractopamine treated cattle, ocular residues in turkeys were greater because residues were clearly present in RCS tissue of turkeys treated with 22.5 ppm dietary ractopamine HCl (1.02 mg/kg). However, relative to clenbuterol-treated cattle, retinal tissues of turkeys treated with ractopamine and slaughtered with no withdrawal period, contained lower levels of total residues than did clenbuterol treated cattle that were slaughtered without a withdrawal period (Table 6; Elliott et al., 1993a, b,c). The relatively low levels of ractopamine residue are especially pronounced when one considers the large difference in the doses of ractopamine HCl or clenbuterol administered to the respective groups of animals. Similarly, Gowik et al. (2000) showed that the magnitude of retinal ß-agonist residue in turkeys was compound specific.
Ractopamine Binding to Melanin
In contrast to the apparent low propensity for ractopamine to accumulate in pigmented ocular tissues in vivo, data generated in this study suggest that ractopamine HCl has a high propensity to bind to melanin. Figure 3 shows that the displacement of radiolabeled ractopamine HCl by clenbuterol was less efficient than its displacement with fenoterol or ritodrine. When [14C]ractopamine HCl was displaced by unlabeled ractopamine, the resulting curve (data not shown) was superimposable on the displacement curves generated by ritodrine and fenoterol. Although the affinity of ractopamine for melanin was not measured, it is apparent that molecules containing two phenyl rings (fenoterol, ritodrine) displaced ractopamine more effectively than compounds with only one phenyl ring. Substitution differences on the phenyl rings of the various ß-adrenergic agonists did not seem to have a great effect on the displacement of ractopamine from melanin.
Accumulation of drugs in ocular tissues is dependent upon the chemistry of the drug in question (Leinweber, 1991). Generally, polycyclic compounds with coplanar fused ring structures, steroids, and hydrophobic primary amines are good candidates for ocular accumulation, especially when the accumulation is specific to or for the pigmented tissues of the eye (Leinweber, 1991; Ings, 1984). The ubiquitous pigment melanin is believed to be responsible for the accumulation of drugs in the eye, but the exact mechanism of drug binding to melanin is unknown. Melanin is concentrated in the retina and choroid tissues (Guyton, 1986). Depending upon the nature of the drug, electronic, steric, and acid/base considerations all seem to influence the degree of binding to melanin (Ings, 1984).
Variations in the propensity of phenethanolamine ß-agonists to bind to melanin in vitro have been documented. For example, Sauer and Anderson (1994) determined that clenbuterol and salmeterol, but not salbutamol, bind strongly to melanin. The phenyl rings attached to the ß-carbons of salmeterol and salbutamol are identical, but salmeterol has an additional phenyl ring attached to the ethanolamine ß-carbon via a long alkyl chain. Thus, the difference in the propensities of the two drugs to bind to melanin is thought to be due to differences in hydrophobicity. Howells et al. (1994) determined that melanin binds more clenbuterol (73% of that added) than salbutamol (23% of that added) when equimolar concentrations were added to a suspension of melanin. These data suggest that phenyl group substitutions have major effects on the propensity of ß-agonists to bind to melanin. These results are supported by data of Sauer and Anderson (1994) who also showed that salmeterol and clenbuterol bind to melanin to a greater extent than salbutamol. Salbutamol is often supplied as a hemisulfate salt rather than an HCl or HBr salt, but whether this would influence the in vitro binding of salbutamol to melanin is not known.
In the case of ractopamine, data from this study clearly show that when ractopamine is glucuronidated, the propensity of the molecule to bind to melanin is virtually eliminated. Because ractopamine is glucuronidated at either phenol, one could speculate that glucuronidation disrupts the interaction of ractopamine’s phenol groups with melanin and that this interaction must be crucial for binding. Data from this study support this supposition to some extent because ß-agonists with two phenyl groups displaced [14C]ractopamine from melanin more efficiently than ß-agonists with only one phenyl group. Glucuronidation of ractopamine also causes the formation of a zwitterionic species at pH 7.4 with a cationic secondary amine and an anionic C-6' carboxyl on the glucuronic acid moiety. Glucuronidation, therefore, could also disrupt ionic interactions between melanin and ractopamine.
In vivo, residues of ractopamine in ocular tissues of cattle and turkeys were less than might be expected when compared to clenbuterol and, perhaps, other ß-agonists (Gowik et al., 2000). This is especially true when one considers the propensity that ractopamine HCl has for binding to melanin in vitro, and the relatively large doses of ractopamine administered to cattle and turkeys in the present studies. Malucelli et al. (1994) measured residues of parent clenbuterol, salbutamol, and terbutaline in the eyes of broiler chickens that had been fed 1, 10, and 10 ppm of each drug, respectively, for 14 consecutive days. Residues of clenbuterol in the eyes of the birds at zero withdrawal were 90 ppb; residues of salbutamol and terbutaline in the eyes at zero withdrawal were 85 and 22 ppb, respectively. Given the 10-fold differences in dose, these data indicate that after in vivo administration clenbuterol has a greater propensity to bind to ocular tissues than either salbutamol or terbutaline. In addition, ractopamine dosed at 7.5 ppm for 7 d resulted in no detectable residues in dissected ocular tissues of turkeys. These results, in conjunction with results from the cattle study, suggest that the propensity of ractopamine HCl to bind to ocular tissues of turkeys is likely less than that for clenbuterol and salbutamol. Ractopamine administration to turkeys at higher doses will cause some accumulation of the drug in ocular tissues, however.
Differences between the propensity for ractopamine to bind to melanin in vitro and the relatively low residues which are present in ocular tissues after the in vivo administration of ractopamine HCl may be due to the rapid metabolism of ractopamine to ractopamine glucuronides and their subsequent excretion. Smith et al. (1993), showed that turkeys excreted approximately 94% of an oral dose (6.7 mg/kg BW of [14C]ractopamine HCl) within 48 h of administration. The major portion of this radioactivity (89%) was excreted in the first 16 h after administration. Analysis of urine from the turkeys showed that only about 8% of the urinary radioactivity (4.2% of the total dose) was parent ractopamine. The other major route of ractopamine excretion by turkeys is via the bile, and little to no parent ractopamine is present in bile (Smith et al., 2000). Thus, parent ractopamine does not remain circulating for extended periods of time. This is in contrast to ß-agonists, such as clenbuterol, which has an extended plasma half-life in species such as cattle (Meyer and Rinke, 1991), rats, rabbits, and humans (Yamamoto et al., 1985) and which is excreted to a large extent as unmetabolized parent compound (Morgan, 1990). Major differences in the metabolism and disposition of ß-adrenergic agonists in farm animals have been discussed by Smith (1998).
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Implications
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These data provide evidence that residues of ractopamine HCl do not accumulate in pigmented ocular tissues of cattle and turkeys to the degree that residues of other ß-agonists accumulate in ocular tissues of livestock species. These results suggest that ocular tissues may not be as useful for monitoring animals for exposure to ractopamine as ocular tissues of animals exposed to ß-agonists such as clenbuterol. If ocular tissues are used to determine ractopamine exposure, the analytical method used would need to have sensitivities normally provided with immunoassays or mass spectrometry.
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Footnotes
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1 The technical assistance of George Bewley and Matthew McGuffey is greatly appreciated. 
2 Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.
3 Current Address: USDA/ARS, Biosciences Research Laboratory, P.O. Box 5674, University Station Fargo, ND 58105-5674.
4 Retired.
Received for publication November 20, 2001.
Accepted for publication May 30, 2002.
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Literature Cited
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Abstract
Introduction
