The distribution and excretion of zinc bacitracin-14C in rats and swine

The distribution and excretion of zinc bacitracin-14C in rats and swine

TOXICOLOGY AND APPLIED PHARMACOLOGY17,366-374 The Distribution Bacitracin-14C (1970) and Excretion of Zinc in Rats and Swine JULIAN DONOSO, GERA...

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TOXICOLOGY

AND APPLIED PHARMACOLOGY17,366-374

The

Distribution Bacitracin-14C

(1970)

and Excretion of Zinc in Rats and Swine

JULIAN DONOSO, GERALD 0. CRAIG,

AND

ROBERT S. BALDWIN

Woodard Research Corporation, Herndon, Virginia 22070; and Research Department, Commercial Solvents Corporation, Terre Haute, Received

Indiana

June 17, 1969

The Distribution and Excretion of Zinc Bacitracin-14C in Rats and Swine. JULIAN, CRAIG, GERALD O., and BALDWIN, ROBERT S., (1970). Appl. Pharmacol. 17, 366-374. The biosynthesis and preparation of zinc bacitracin-14C are described. Zinc bacitracin-14C was administered

DONOSO, Toxicol.

po to rats and swine, and the distribution of radioactivity in excreta and tissues was determined by liquid scintillation counting. In both species, radioactivity was found almost exclusively (92-93 %) in the feces; minimal radioactivity was found in both urine and blood of swine. Total recovery of

the administered dose was 98 % for rats and 95 % for swine. Analysis of the data indicates that zinc bacitracin wasnot significantly absorbed by rats or swine following oral ingestion.

Zinc bacitracin is a widely used antibiotic for supplementing animal feed rations to improve weight gain and feed efficiency and to reduce disease of livestock. Although the efficacy of the bacitracin has been well established since it was first described by Johnson et al. (1945), little is known concerning its metabolism and fate in the animal. Previous studies have shown that bacitracin is not assimilated significantly into the circulating body fluids after oral ingestion of the compound in doses up to 5000 units/kg daily, as determined by microbiological assay (Payne et al., 1951). Recovery studies in dogs accounted for less than 5 % of the po administered dose of bacitracin (Scudi et al., 1947). Using a modification of Craig’s (1965) microbiological assay procedure for low-level bacitracin assay, J. Mitchell (1967, personal communication) found that 26-34x of the zinc bacitracin administered po to rats was recovered in the feces. Zinc bacitracin was administered in a diet containing 500 g of zinc bacitracin per ton of feed. These studies would indicate that in animals receiving less than 5000 units/kg of body weight, a considerable portion of the bacitracin activity was destroyed in the intestinal tract and that no significant quantities of the drug were detected in the urine. (The sensitivity of the newer microbiological assay procedures is of the order of 24&720 ppb.) Since microbiological assay methods have proved inadequate in fate and distribution studies and have failed to account for a significant amount of the administered dose, metabolic studies with isotopically labeled bacitracin appeared warranted. METHODS

Zinc bacitracin-14C, uniformly labeled (u.l.), was prepared biosynthetically using a bacitracin-producing strain of Bacillus licheniformis and employing D-gh.ICOSe-'4C (u.1.) or an amino acid-14C (u.1.) mixture as the sources of the label, as described below. 366

ORAL

ZINC

367

BACITRACIN-14C

A two-stage inoculum was developed by introducing spores into a 300-ml conical flask containing 60 ml of a sterile medium consisting of 1% NZ [email protected] A, 0.25 % beef extract (Difco), 0.005 % lard oil, and tap water (pH 7.2). The flasks were incubated at 32-35°C for 24 hr on a rotary shaker. The second-stage development was prepared in a similar manner using 7 % by volume of the first-stage cells as inoculum, and incubating for 15 hr. Seven hundred milliliters of a sterile production medium (soybean meal flour, 4 %; starch, 1 ‘A; CaCo3, 0.65 ‘A; MgS04*7H,0, 0.025 ‘A; lard oil, 0.003 ‘A; and tap water, pH 7.1-7.4) in a l-liter automatically stirred fermentation vessel, was inoculated with 7 % by volume of the second-stage inoculum described above. Sterile air was introduced at 9.3 1per liter of medium, per minute, and agitation was adjusted to obtain a dissolved oxygen tension of 2-7 ppm as determined by an oxygen analyzer(Beckman Instruments). At 6 hr after inoculation, 90 &i of D-glucose-‘4C (u.1.) or 90 &I of amino acid-r4C (u.1.) mixture was introduced into the medium. At 24 hr the biosynthesis was terminated, and the bacitracin was recovered by zinc precipitation by a modification of the procedure of Chornock (1952) and of Zinn and Chornock (1958). Repeated washing of the zinc bacitracin precipitate was carried out until the washings were devoid of radioactivity. The precipitates were transferred to 20 ml serum vials, lyophilized, sealed, and stored at 5°C until used. Analysis of the products for purity and homogeneity was conducted using thin-layer chromatography, antimicrobial spectrum identification procedure, ultraviolet absorption spectrometry, and microbiological assay. Analytical data are summarized in Table 1. TABLE CHEMICOBIOLOGICAL

Property Label used Potency” Zinc TLCb TLC radiometric Specific activity Ultraviolet absorption

PROPERTIES

Rat study D-Glucose-14C (u.1.) 74.1 units/mg 5.23 % R, 0.375single spot RI 0.375single spot 5.89 x 1O-4 &i/mg 253 nm (max)”

1 OF ZINC

BACITRACIN-14C

Swine study L-Amino acid-Y (u.1.) 64.1 units/mg 6.11% Rf 0.370single spot Rf 0.370single spot 0.89 x 1O-3 $i/mg 253 nm (max)”

a USP. b Thin-layer chromatography; butanol-acetic acid-H,O, 4: 1: 1. ’ Purchased from New England Nuclear Corporation, Boston, Massachusetts. d Methanol, 1 cm path length.

The 638 mg of product used for the rat study was produced from D-glucose-r4C (u.1.) and assayed 74.1 units/mg. The product prepared for the swine study consisted of 2.735 g of bacitracin-14C produced from amino acid- r4C (u.1.) mixture, and coprecipitated as zinc bacitracin with 150 g of unlabeled commercial bacitracin to yield 4.235 g of zinc bacitracin, assaying 64.1 units/mg. The 2 products produced, equaled, or exceeded the specifications of commercial grade zinc bacitracin.

DONOSO, CRAIG, AND BALDWIN

368

Rats. Three male albino rats (Charles River CD strain) weighing 200-250 g were used 7 days after arrival in the laboratory. Theseanimals were placed in modified Roth all-glass metabolism cages for an acclimation period of 4 days prior to starting the experiment. The rats were then dosed by stomach tube for 4 consecutive days with an aqueous suspensionof zinc bacitracin-t4C at the rate of 20-25 mg/kg day (Table 2). TABLE 2 ZINCBACITRACIN-'VTOTALDOSAGEPERANIMAL

Animal species

Body weight (8)

Rat 1 Rat 2 Rat 3

243 292 255

Pig 1

19,846

Pig 2 Pig 3

19,850 19,971

Zinc bacitracin-14C (administered/kg/day) m

Units

25.1 20.5 22.8 10.4 12.0 11.4

1860 1519 1689 660 762 730

The experimental period lasted 96 hr after the initial dose. During this time, collections of feces, urine, and expired CO1 were made at 24,48, 72, and 96 hr. The animals were sacrificed by decapitation 24 hr after the final dose. The liver, kidneys, hindlimb muscle, mesenteric fat, brain, skin, and gastrointestinal tract were removed from each rat and frozen prior to radioactivity determination. Blood was also collected, and the plasma was refrigerated until radioanalysis. Swine. Three male pigs, each weighing approximately 20 kg, were obtained locally and acclimated in the laboratory for 1 week. Each animal was placed in a plexiglass metabolism cage(Craig and Donoso, 1968),specifically designedfor this experiment, for 4 days before administration of the radioactive compound. The pigs were fed for 3 consecutive days with a standard diet containing 252 ppm of zinc bacitracin-14C (Table 2). The food mixture was presented several times each day in small portions in order to minimize loss of the radioactive compound or possible contamination of the cage by dispersion of food. The total experimental period was 96 hr, with collections of urine, feces, and COz at 24, 48, 72, and 96 hr. At termination of this period the animals were sacrificed by injection of sodium pentobarbital and subsequentdecapitation. Liver, kidneys, hindleg muscle, mesenteric fat, brain, skin, and gastrointestinal tract were removed from each pig and frozen prior to radioanalysis. Blood was collected, and the plasma was refrigerated until analyzed. Blood levels. Male albino rats (Charles River CD strain) were acclimated to laboratory conditions for 2 weeks. The animals were then dosed by oral intubation with 73 mg/kg zinc bacitracin- 14Chaving specific activity of 9.89 x 10e3&i /mg or an equivalent of about IO5 cpm per animal. The experimental period lasted for 24 hr with bile and blood collections at l/2, 1,2, 4,7, 17, and 24 hr after dosing. At each time interval, one animal was anesthetized with ether and the thoracic cavity was opened. Blood was obtained by heart puncture, and

ORAL

ZINC

BACITRACIN-14C

369

bile was collected by drainage of the common bile duct, prior to sacrificing the animal. Blood was allowed to clot; it was then centrifuged, and the serum was removed. All samples were refrigerated until radioanalysis. Radioanalysis. Radioactivity assays were made in a Nuclear Chicago No. 6801 liquid scintillation spectrometer, using toluene-14C as the calibration standard. The scintillation solution used contained 1.0 % PPO (2,5-diphenyloxazole), 0.5 % POPOP [1,4-bis-2-(4-methyl-5-phenyloxazolyl)benzene], and 5.0 y0 naphthalene, in a 5 : 1 mixture ofp-dioxane and ethylene gycol. All samples were counted in 20-ml glass vials. Feces and intestinal contents were homogenized, dried at 45°C and ground to a fine powder. Approximately l-g aliquots were placed in small cellophane envelopes, and 0.2 ml of 10 % sucrose solution was added to each sample to provide additional fuel for the combustion process. The envelopes were then dried under an infrared lamp and combusted in Thomas-Lisk oxygen-combustion flasks. A solution of 40% 2-phenylethylamine in methanol was added through the sidearm after combustion to absorb the t4C02 produced, and appropriate aliquots of this solution were then transferred to counting vials containing scintillation solution. Tissues were homogenized and representative aliquots were weighed into cellophane envelopes; these, in turn, were dried and combusted as previously described. The efficiency of the combustion technique was determined by adding zinc bacitracin-14C standard to several fecal and tissue samples before combustion. The recovery was 93 %. All combusted samples were counted three times, for 20 min each time, and then recounted after the addition of zinc bacitracin-14C internal standard, in order to obtain quenching corrections for each particular sample. Plasma, urine, and bile were added directly to counting vials containing scintillator solution and counted for 20 min, then recounted after the addition of internal standard. Serum samples (0.4 ml) were pipetted into counting vials and digested with 2 ml Hyamine hydroxide. All digestions were performed in duplicate for 24 hr at room temperature. After digestion had ceased, the counting vials were filled with 15 ml of scintillator solution and counted as described. Expired carbon dioxide was collected during the experimental periods by means of absorption towers containing a solution of 2-phenylethylamine, methanol, and distilled water. These towers were drained at the indicated time intervals, and the absorbing solutions were saved for radioanalysis. Sample aliquots were transferred to counting vials containing scintillator solution, counted, and corrected for quenching by addition of internal standard. The technique of internal standardization was used to determine the quenching and percent recovery of each sample. Sample counts were subjected to statistical analysis in order to evaluate their significance as compared to background counts. The final calculations were expressed as the amount of zinc bacitracin-r4C found. RESULTS Absorption

and Distribution

Throughout the experimental periods no abnormal signs were observed in any of the animals. Eating habits, urine, and feces of each rat or pig appeared normal, as did the organs of each animal at autopsy.

3

2

Total

Total

24 48 72 96

24 48 72 96

24 48 72 96

1

Total

Hours after dose

Animal No.

10.7 -

10.7

2.5

1.4 6.4 8.8 4.1 20.7

1.1 1.1 6.6 13.6 22.4

1.8 6.8 9.3 5.1 23.0

-

Mg

Feces

EXCRETION

% Dose

Rats

BACITRACIN

2.5 -

-

-

Mg ~__~ -

Urine

ZINC

TABLE IN RATS

6.2 27.5 37.6 17.5 88.8

4.5 4.4 27.5 56.8 93.2

7.2 27.8 38.1 20.9 94.0

‘A Dose

3

0.9 2.2 6.1 5.2 14.4

4.8 4.9 9.7 4.7 4.4 1.3 10.4

Mg

AND SWINE

Urine

0.1 0.3 0.9 0.8 2.1

0.8 0.8 1.6 0.7 0.6 0.2 1.5

% Dose

Swine

1.3 11.1 19.7 23.3 55.4 10.9 7.5 35.9 54.3

74.4 51.7 245.6 371.7

2.3 10.4 37.4 50.1

% Dose

9.4 79.0 104.6 167.0 396.0

14.3 64.6 231.9 310.8

Mg

Feces

f

g g G

B P “5

i3 3 3

ORAL

ZINC

371

BACITRACIN-14C

The concentrations of radioactivity in tissues and excreta obtained from rats and swine at various times after po administration of zinc bacitracinJ4C are presented in Tables 3-5. In both species the compound was either excreted in the feces or retained in the gastrointestinal tract, thus indicating that zinc bacitracin-14C was not readily absorbed. In rats, most of the 14C label was found in the feces, primarily within 24 hr after administration (Table 3). With the exception of one animal, no activity was found in the urine. This animal showed about 10 y0 of the total dose present in the 24-hr urine sample following the first dose. However, this amount is equivalent to 43 % of the first dose, and is most certainly due to contamination of the urine, probably by regurgitation of the dose suspension. As shown in Table 4, no radioactivity was found in any tissues or expired carbon dioxide, and only about 2 ‘A of the total dose remained in the gastrointestinal tract. The total mean recovery of the administered dose was 98 %. TABLE

4

DISTRIBUTION OF ZINC BACITRACINJ~C IN RAT TISSUES ANDEXCRETA

Rat No. 1

Zinc”

Rat No. 2

Rat No. 3

Zinc

bacitracin 6-m)

% Dose

bacitracin @d

% Dose

GI Tract

NDb 23.0 ND ND 0.7

94.3 2.8

ND 22.4 ND ND 0.7

93.2 2.9

Total Total Dose Percentrecovery

23.7 24.4 97.1

Sample

Urine Feces co2 Tissues

23.1 24.0 96.2

-

Zinc bacitracin 6-w)

% Dose

2.5” 20.7 ND ND 0.3

10.7 88.8 1.3

23.5 23.3 100.8

aSensitivity: 1.6ppm. bNo detectableradioactivityat 5% confidencelevels. cFirst 24-hrsample only; equivalentto 43%of first day’sdose(10.7%of ~tul dose).Contamination wasprobablyresponsible.

In swine, the distribution of 14C radioactivity followed a pattern similar to that in rats, although larger concentrations were found in the gastrointestinal tract, particularly the large intestine (Table 5). However, unlike the rats, swine exhibited small amounts of 14C radioactivity in the urine (average of 1.7 y0 total dose). Although it is conceivable that some of this radioactivity might originate from contamination, the urine values closely correlate with those obtained from plasma (approximately 1.7 y0 average, based on body weight) and suggest minimal absorption of the compound. Again, no activity was found in tissues or expired carbon dioxide. The total mean recovery for swine was 95 %.

372

DONOSO,

CRAIG,

AND

BALDWIN

TABLE 5 DISTRIBUTION

OF ZINC

BACITRACIN-‘“C

IN SWINE

Swine No. I

Sample __~.___~~ Urine Feces co2 Tissues Plasmac Large intestine Small intestinestomach Total Total Dose Percent recovery

~~

TISSUES AND EXCRETA

Swine No. 2

Swine No. 3

Zinc” bacitracin b-a)

“//, Dose

Zinc bacitracin Wit)

“4 Dose

Zinc bacitracin (mg)

7: Dose

9.7 310.8 NDb ND 9.3 252.1

1.6 50.1 1.5 40.6

10.4 396.0 ND ND 10.6 307.3

1.5 55.4 1.5 43.1

14.4 371.1 ND ND 14.9 214.3

2.1 54.3 2.2 31.4

2.2

0.3

4.3

0.7

0.5

0.1

583.1 619.2 94.3

728.6 714.6 102.0

615.7 683.0 90.1

0 Sensitivity: 1.0 ppm. b No detectable radioactivity at 5% confidence levels. c Calculated on the basis of 5% of body weight (20 kg).

Blood Concentrations Measurements of radioactivity in serum obtained from rats at various times following single oral doses of zinc bacitracin- 14C are summarized in Table 6. The serum content of 14C remained almost constant throughout the 24-hr experimental period. By the end of the first 30 min 1.33 % of the administered 14C was found in the serum, compared with 0.60% of the administered dose at 24 hr. No activity was found TABLE

6

CONCENTRATION OF ‘T RADIOACTIVITY IN SERUM OF RATS AT VARIOUS TIMESAFTER A SINGLE ORAL DOSE OF ZINC BACITRACIN-I’%

Time (hr)

Percent of dose in blood volume”

0.5 1 2 4 7 17 24

1.33 0.68 0.86 1.35 0.92 1.61 0.60

0 Calculated as 5% body weight.

ORAL ZINC BACITRACIN-14C

373

in any bile samples. These results suggest that although some absorption of zinc bacitracin-14C does occur, the high fecal recovery is due to the unabsorbed compound, and is not attributable to enterohepatic recirculation. DISCUSSION

Bacitracin is an antibiotic polypeptide active against gram-positive bacteria, and is commonly used as a growth factor for swine, chickens, and turkeys. Thus, it may be expected that the compound would undergo hydrolytic cleavage of the peptide bonds in the gastrointestinal tract, with absorption and metabolism of the split products. However, the results obtained in our experiments indicate that zinc bacitracin is not significantly absorbed by rats or swine following oral ingestion. This finding is in agreement with that reported by Scudi ef al. (1947) and Longacre and Waters (1951), who investigated the absorption and excretion of bacitracin in dogs and man. These investigators suggested that orally administered bacitracin might remain as bacitracin and be absorbed into the body fluids at an insignificant rate or that it might be metabolized, or fixed in the intestinal tract. Their suggestions were based on results obtained by microbiological assays of urine, blood, and feces. Our studies show that although there appears to be a fairly constant, minimal absorption of zinc bacitracin-14C, as evidenced by the small amounts of radioactivity found in both the urine and blood of swine, most of the radioactivity is recovered in the feces and intestinal contents. Unfortunately, during the absorption and distribution studies it was technically impossible to monitor blood levels throughout the experimental periods. Thus it can be speculated that the compound is absorbed and recycled via enterohepatic circulation into the small intestine, either unmodified or metabolized, which would account for the high fecal recovery. However, the 14C blood levels in rats following a single oral dose of zinc bacitracin showed a constant, almost uniform serum 14C content. Furthermore, no detectable 14C activity was found in the bile. These findings suggest that the bacitracin is degraded to some extent in the gastrointestinal tract, but that only a small amount of the split products is absorbed, w-hile most of the administered dose is eliminated in the feces. It may be noticed here that in the rat absorption experiment, no blood residues were reported. This discrepancy may be explained by the fact that the specific activity of the administered zinc bacitracin 14C was much lower; thus the blood 14C concentrations were not detectable. It remains to be determined whether the fecal 14C content is as unchanged zinc bacitracin or a metabolite. Experiments are currently in progress in an attempt to identify the source of fecal radioactivity. ACKNOWLEDGMENT

The authors wish to expresstheir gratitude to Mr. J. Dickerson for skilful technical assistance in preparation of bacitracin by biosynthesis. REFERENCES

CHORNOCK, F. W. (1952).CommercialSolventsCorporation, Zinc bacitracin feed supplement, U.S. Patent 2,809,892. CRAIG,G. H. (1965).Microbiological assayfor bacitracin in animalfeed.J. Assoc.Ofic. Agr. Chemists48,256-264.

374

DONOSO, CRAIG, AND BALDWIN

CRAIG, G. O., and DONOSO, J. (1968). A metabolism cage designed for radioisotope studies with pigs. Capsules 9, 1, Woodard ResearchCorporation, Herndon, Virginia. JOHNSON, B. A., ANKER,H., and MELENEY, F. L. (1954).Bacitracin, a new antibiotic produced by a memberof the Bacillus subtilis group. Science 102, 316. LONGACRE, A. B., and WATERS,R. M. (1951).Observationson absorptionof bacitracin blood levelsfollowing oral administration. Scrug.Gynecol. Obstet. 92, 213. PAYNE,H. G., SPENCER, J. N., and SCHULTZ,F. H. (1951).The oral toxicity of bacitracin. Antibiot. Chemotherapy 1, 387-391. SCUDI,J. V., CLIFT,M. E., and KRUGER,R. A. (1947).Somepharmacologicalcharacteristics of bacitracin. II. Absorption and excretion of bacitracin in the dog. Proc. Sot. Exptl. Biol. Med. 65, 9-13, 1967. ZINN, E., and CHORNOCK, F. W. (1958).Bacitracin, CommercialSolvents Corporation, U.S. Patent 2,834,711.