Identification of turkey biliary metabolites of ractopamine hydrochloride and the metabolism and distribution of synthetic [14C]ractopamine glucuronides in the turkey.

1. The bile duct cannulated turkey poult (n = 3) dosed orally with [14C]ractopamine HCl [(1R*,3R*),(1R*,3S*)-4-hydroxy-alpha-[[[3-(4-hydroxy[14C]phenyl)-1-methy lpropyl]amino]methyl]-benzenemethanol hydrochloride; 19.9 mg; 9.28 microCi] excreted 37.4 +/- 12.1% (mean +/- SD) of the administered radioactivity in bile by 24 h post-dosing. 2. A mono-glucuronide, conjugated at C-10 (the methylpropylamino phenol) of ractopamine, accounted for 76.6% of biliary radioactivity. 3. Urine collected from the colostomized turkey poult (n = 3) orally dosed with synthetic [14C]ractopamine-glucuronides (10.1 mg; 3.6 microCi) contained 11.9 +/- 1.0% (mean +/- SD) of the administered radioactivity 24 h after dosing, indicating that some absorption of radioactivity occurred. Faeces contained 60.6% of the administered radioactivity and carcasses (with gastrointestinal tracts) contained 23.3% of the starting radioactivity. 4. Five colostomized poults were fitted with bile duct cannulas and were dosed intraduodenally with 10.2 mg (3.6 microCi) synthetic [14C]ractopamine-glucuronides. Urine and bile contained 15.5 +/- 2.2 and 16.8 +/- 2.1% respectively of the administered radiocarbon by 24 h post-dosing. Faeces contained 54.3% of the administered radioactivity. Total absorption of the dosed radioactivity averaged 33.4%. 5. Bile and urine collected from the colostomized, bile-duct cannulated bird contained mainly ractopamine glucuronides. Indirect evidence suggests that the dosed ractopamine glucuronides were not absorbed intact.

Distribution of radiocarbon after intramammary, intrauterine, or ocular treatment of lactating cows with carbon-14 nitrofurazone.

Three lactating Holstein cows (634 to 698 kg) were dosed, respectively, with 65.6 mg (44.5 microCi/mg), 131.2 mg (20.1 microCi/mg), or 8.4 mg (141.3 microCi/mg) of [14C]nitrofurazone by intramammary, intrauterine, or topical ocular administration. Intramammary and intrauterine treatments were single doses; ocular treatment was daily for 4 consecutive d (2.1 mg/d). Cows were slaughtered after 72-h withdrawal periods. Excreta and milk were quantitatively collected from each cow after dosing. Seventy-two hours after treatment, urine, feces, and milk contained 62.9, 17.6, and 2.3%, respectively, of the radiocarbon administered intramammarily to the cow. Radioactive residues in milk collected from the dosed quarter were 150 ppb (nitrofurazone equivalents) and were 39 ppb in milk collected from the undosed quarters at 12 h after dosing. Urine, feces, and milk from the cow that received the intrauterine dose contained 12.24, 5.17, and 0.13% of the administered dose, respectively, at 72 h after treatment. Concentrations of total radioactive residues in milk were 9.3 ppb at 12 h after dosing. For the cow that was dosed ocularly, the cumulative excretion of radiocarbon in urine, feces, and milk was 17.6, 28.5, and 0.5% of the dose, respectively. Milk residues from the cow that was dosed ocularly were never > 1 ppb of nitrofurazone equivalents. Livers and kidneys contained the greatest amounts of residues relative to other edible tissues. Parent nitrofurazone was not suitable as a marker compound to determine total residues in milk using HPLC analysis. Radioactive residues were available systemically and were excreted in milk after intramammary, intrauterine, or ocular application of [14C]nitrofurazone. Illegal residues in milk and edible tissues would result from the administration of nitrofurazone to lactating cows.

Distribution, elimination, and residues of [14C]clenbuterol HCl in Holstein calves.

Clenbuterol HCl is a beta-adrenergic agonist that has been used illegally in Europe and the United States by some livestock producers to increase carcass leanness. The objectives of this study were to determine the metabolic disposition, distribution of radioactivity, and the concentrations of parent clenbuterol in tissues after a single oral dose of [14C] clenbuterol HCl in calves. [14C]Clenbuterol HCl (1.59 microCi/mg, 3 mg/kg BW) was administered to a 74- and a 96-kg Holstein bull calf as a single oral dose. Blood samples were taken at 1, 3, 6, 12, 24, 36, and 48 h after dosing; urine and feces were collected separately and placed into respective pools from 0 to 6, 6 to 12, 12 to 24, 24 to 36, and 36 to 48 h after dosing. At 48 h after dosing, calves were anesthetized and exsanguinated, and visceral organs, bile, eyes, brain, skeletal muscle, skin, adipose tissue, and the remainder of the carcass were collected. Tissues were processed by coarse grinding through a Hobart grinder, followed by homogenization on dry ice. Eyes were dissected and the various tissues and excreta were assayed for total radiocarbon content by combustion analysis and(or) liquid scintillation counting. Parent clenbuterol was measured in selected tissues by HPLC after solvent extraction. Urinary, fecal, and carcass radioactivity averaged 41.5 +/- 8.1, 2.4 +/- .4, and 52.3 +/- 8.7% of the dose, respectively (mean +/- SD.). Average total recovery of radiocarbon was 96.2 +/- .4%. Radioactive residues present in carcasses averaged (ppm; mean +/- SD.): blood, .6 +/- .2; heart, 1.4 +/- .0; lungs, 8.4 +/- 1.7; spleen, 2.6 +/- .3; liver, 5.0 +/- .4; kidney, 5.9 +/- .0; brain, 1.9 +/- .4; adipose tissue, 1.1 +/- .2; rumen, reticulum, omasum, abomasum, 2.3 +/- .4; small intestine, 3.2 +/- .3; large intestine, 4.0 +/- .4; skeletal muscle, 1.0 +/- .2; bile, 12.5 +/- 4.0; white skin, .7 +/- .1; black skin, 4.0 +/- .7; remainder of the carcass, 1.0 +/- .2. Ocular residues were as follows: aqueous humor, 6.3 +/- 1.2; cornea, 13.5 +/- 8.6; iris, 255.8 +/- 167.0; lens, 2.3 +/- 1.5; vitreous humor, 2.2 +/- .4; retina/choroid, 84.5 +/- 34.1; sclera, 11.1 +/- 2.1. Mean concentrations of parent clenbuterol in tissues were (ppm; mean +/- SD): lung, 6.8 +/- .9; liver, 2.2 +/- .5; kidney, 3.7 +/- .5; and heart, .9 +/- .1. Parent clenbuterol represented from 43.9% of the total residue in liver to 81.2% of the total residue in lung.

Metabolism of 14C-sulphadimethoxane in swine.

1. 14C-sulphadimethoxine (4-amino-N-(2,6-dimethoxy-4-pyrimidinyl)benzene-[U-14C]-sulphonamide; 14C-SDM) was given orally (60 mg/kg body weight) to eight swine (weight 27-32 kg). Urine and faeces were collected from 0 to 72 h after dosing and tissue samples were collected from animals exsanguinated at 12, 24, 48 and 72 h after dosing. The concentration of total 14C-labelled residues (14C-SDM equivalents) in tissues other than the gastrointestinal tract ranged from 99-1 ppm (plasma) to 13.8 ppm (adipose tissue) 12 h after dosing. Seventy-two hours after dosing tissue concentrations ranged from 5.4 ppm (plasma) to 0.5 ppm (skeletal muscle). The concentration in the large intestine was substantially higher (10.4 ppm) than in the stomach (2.8 ppm) and small intestine (1.4 ppm) 72 h after dosing. 2. Of the 14C, 77% was excreted in the urine from 0 to 72 h after dosing with 14C-SDM, mostly in the 0-24-h collection. Fifteen percent was excreted in the faeces from 0 to 72 h after dosing, with most of this occurring 36-72 h post-dosing. 3. 14C-SDM accounted for 24% (liver) to 66% (adipose tissue) and the N4-acetyl derivative of SDM (N4-Ac-SDM) accounted for 10% (skeletal muscle) to 35% (kidney) of the total 14C in the tissues 12 h after dosing. The N4-glucose conjugate of SDM (G-SDM) was a major 14C-labelled compound in skeletal muscle (21% of total) and liver (28%) but it was not detected in adipose tissue or kidney. The N4-glucuronic acid conjugate of SDM (GA-SDM) was a minor metabolite in kidney, but was not detected in other tissues collected 12 h after dosing. Desamino SDM was a minor metabolite in the kidney. A minor metabolite in plasma was identified as the sulphate ester of 3-hydroxysulphadimethoxine. 4. 14C-labelled fractions isolated from 0 to 6-h urine included N4-Ac-SDM (82%), SDM (3%) and GA-SDM (6%).

The disposition of 14C-levamisole in the lactating cow.

1. 14C-Levamisole 1(-)-2,3,5,6-tetrahydro-6-phenyl[U-14C]imidazo[2,1-b]-thiazole was administered orally and subcutaneously to lactating cows (8 mg/kg body weight). Urine, faeces, milk and blood samples were collected from 0-48 h after dosing and tissues were collected 48 h after dosing. 2. 14C-Labelled residues (ppm 14C-levamisole equivalents) in blood were highest at 3 h (2.2 ppm, oral dose) or 6 h (2.1 ppm, subcutaneous dose) and then declined to less than 0.5 ppm 48 h after dosing. 3. 14C-Labelled residues in milk were highest in samples collected from 0-12 h after dosing (1.55 ppm and 1.86 ppm of levamisole equivalents from oral and subcutaneously dosed animals, respectively) and declined to 0.06 ppm in milk collected from 36-48 h after dosing. Milk collected from 0-48 h after dosing accounted for 0.2% (oral dose) and 0.6% (subcutaneous dose) of the total 14C-activity administered as 14C-levamisole. The parent compound, 14C-levamisole, accounted for 12% or less (declined with time after dosing) of the total 14C-activity in the milk. Three 14C-labelled metabolites (formed by oxidation of imidazoline ring and/or opening of thiazolidine ring) in the milk were isolated and identified. 4. Urinary excretion accounted for 83% and 84% and faecal excretion accounted for 11% and 9% of the total 14C-activity given orally and subcutaneously, respectively, as 14C-levamisole. No 14C-levamisole was detected in the urine; the major urinary metabolite (formed by opening of thiazolidine ring) was isolated and identified. 5. The 14C-activity remaining in the animals 48 h after dosing was widely distributed in body tissues; however, the concentration in the liver was substantially higher than in all other tissues examined. Less than 5% of the 14C-activity in the liver was present as 14C-levamisole.

Identification of ractopamine hydrochloride metabolites excreted in rat bile.

1. Rats dosed orally with 2.85 +/- 0.30 mg [14C]ractopamine HC1 [(1R*, 3R*), (1R*, 3S*)-4-hydroxy-alpha-[[[3-(4-hydroxyphenyl)- 1-methylpropyl]-amino]-methyl]([U-14C]benzenemethanol)hydrochloride] containing 1.44 +/- 0.15 microCi radioactivity excreted 58 +/- 7% of the administered radioactivity in the bile within 24 h. Absorption and excretion of radioactivity was rapid as 55% of the administered radiocarbon was excreted into the bile during the first 8-h collection period. 2. Radioactivity excreted in rat bile was partitioned by XAD-2 column chromatography and reverse-phase hplc into at least seven different crude metabolite fractions; metabolites representing approximately 76% of the biliary radioactivity were isolated and identified from four of the crude metabolite fractions. 3. Approximately 46% of the biliary radioactivity was identified as a sulphate-ester, glucuronic acid diconjugate of ractopamine. Identification was based on 1H-nmr and negative-ion FAB-ms spectroscopy. Enzymatic and chemical hydrolysis of the sulphate-ester followed by co-chromatography of the hydrolysis products with synthetic ractopamine mono-glucuronides, established the site of sulphation at the C-10' phenol (phenol attached to carbinol) and glucuronidation at the C-10 phenol (phenol attached to methylpropyl amine) of ractopamine. 4. A metabolite representing approximately 6% of the biliary radioactivity was identified as a ractopamine mono-sulphate conjugate by using mass spectral and 1H-nmr techniques. Sulphate was conjugated at the C-10' phenol of ractopamine and was not stereospecific. 5. Approximately 25% of the biliary radioactivity was identified as ractopamine mono-glucuronides. The major site of glucuronidation was at the C-10 phenol, but ractopamine glucuronidated at the C'-10 phenol was also present.

Depletion of residues from milk and blood of cows dosed orally and intravenously with sulfamethazine.

Cows were dosed orally (n = 4) or intravenously (n = 4) with sulfamethazine [sulmet; 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide] for 5 consecutive days (220 mg/kg of body weight on day 1 and 110 mg/kg on days 2-5). The concentrations of sulmet, N4-acetylsulfamethazine (Ac-sulmet), and the N4-lactose conjugate of sulfamethazine (lac-sulmet) were measured in milk and blood collected at 24 h intervals after the last doses of sulmet were given. The method of analysis included (1) spiking of samples with known amounts of 13C6-labeled reference compounds, (2) resolution of the 3 compounds by reversed-phase chromatography, (3) hydrolysis of lacsulmet, (4) treatment with diazomethane to yield N1-methyl derivatives, and (5) gas chromatography/mass spectrometry. The ratios of intensities of selected mass spectral ions containing 12C6 and the corresponding ions containing 13C6 were used for residue quantitation. Sulmet, which was always the most abundant residue in the blood, decreased to less than 100 ppb 4 days after the last doses were given and to less than 10 ppb 7 days after the last doses. The concentrations of sulmet in milk were approximately one fifth the concentrations of sulmet in blood. The concentrations of lac-sulmet and Ac-sulmet in milk were lower than the concentrations of sulmet in milk.

Growth characteristics of rats receiving ractopamine hydrochloride and the metabolic disposition of ractopamine hydrochloride after oral or intraperitoneal administration.

Objectives of this study were 1) to measure the effect of oral or i.p. administration of ractopamine HCl on growth and feed utilization in rats, 2) to determine the total absorption of [14C]ractopamine HCl after oral administration, and 3) to determine the disposition of radioactivity and the urinary elimination of unchanged [14C]ractopamine in rats after oral or i.p. administration of [14C]ractopamine. Twenty-seven female Sprague-Dawley rats (164.6 +/- 5.7 g) were randomly assigned to control (CONT), oral (ORAL), and i.p. (IP) treatments. Control and ORAL rats were implanted i.p with sham pumps, and IP rats were implanted i.p. with osmotic pumps primed to deliver 312 micrograms of ractopamine HCl per 24 h. Control and IP rats received no dietary ractopamine, but ORAL rats received 20 mg of ractopamine HCl/kg of diet. The IP rats had greater cumulative net weight gains and ADG on d 2, 6, 8, 10, and 12 than CONT rats. The ADFI was greater for ORAL rats on d 2 and 4 than for CONT rats, and the gain:feed ratio was greater on d 2, 6, 8, 10, and 12 for IP rats than for CONT rats. Net weight gain, ADG, and gain:feed ratio did not differ between ORAL and CONT rats. Absorption of radioactivity administered orally as [14C]ractopamine (2.9 mg) was 87.9% during a 24-h experimental period; biliary, urinary, and fecal excretion of radioactivity was 58.5%, 28.7%, and 1.4% of that administered, respectively. Urine from rats dosed orally with [14C]ractopamine contained 1.9% of the radioactivity as the parent compound, and urine from rats dosed i.p. contained 22.6% of the radioactivity as parent ractopamine. Ractopamine HCl increased weight gain and efficiency of feed utilization when administered i.p. to rats, but not when administered orally. The ineffectiveness of oral ractopamine for stimulating the growth of rats was probably due to extensive presystemic metabolism of ractopamine.

Metabolism and disposition of ractopamine hydrochloride by turkey poults.

Ractopamine HCl, ((1R*,3R*),(1R*,3S*)-4-hydroxy-alpha-[[[3-(4- hydroxyphenyl)-1-methylpropyl]-amino]methyl]benzenemethanol hydrochloride), is a beta-adrenergic agonist that is under evaluation as a nutrient repartitioning agent in livestock. Because ractopamine metabolism has not been evaluated in turkeys, the objectives of this study were to synthesize and identify products of ractopamine metabolism and to determine the stereoselective metabolism and tissue distribution of [14C]ractopamine HCl in orally dosed turkey poults. Glucuronides of diastereoisomeric [14C]ractopamine, and the (1R,3R) and (1R,3S) stereoisomers of ractopamine were synthesized by use of microsomal proteins immobilized on Sepharose beads. Monoglucuronides conjugated to the phenols at C-10 or C-10' were isolated and identified by 1H-NMR and negative ion FAB/MS. Urine and feces were collected from colostomized turkey poults after oral dosing with 20 mg of [14C]ractopamine HCl (9.28 microCi). Radioactive residues in tissues were highest in the gallbladder and liver. Radioactivity was not detectable in blood 48 hr after dosing and was slightly above background (< 100 dpm/g tissue) in skeletal and cardiac muscle. Urine contained 47.5% of the administered radioactivity by 16 hr after dosing, and by 48 hr 52.0% of the radioactivity was excreted in the urine. Feces contained 36.6% and 41.5% of the dose 16 and 48 hr after dosing, respectively. Unmetabolized ractopamine represented only 8% of the urinary radioactivity; ractopamine glucuronides represented 72% of the urinary radioactivity. Glucuronides conjugated to the C-10 phenol of ractopamine represented 59.8% of the urinary metabolites and were composed of all four ractopamine stereoisomers. Glucuronides conjugated to the C-10' phenol of ractopamine represented 12.7% of the urinary metabolites and were composed of the (1R,3R) and (1R,3S) stereoisomers. Ractopamine was rapidly eliminated from turkeys after oral dosing, and glucuronidation was the major pathway of metabolism. Regioselective glucuronidation occurred favoring the C-10 phenol of ractopamine; glucuronidation of the C-10' phenol of ractopamine was specific for the (1R,3R) and (1R,3S) stereoisomers.

Lactose conjugation of sulphonamide drugs in the lactating dairy cow.

1. 14C-Sulphamethazine (4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene-[U-14C]-sulphonamide; 220 mg/kg of body weight) was given orally or i.v. to lactating dairy cows. Milk collected from 0-48 h after dosing accounted for 2.0% (oral dose) and 1.1% (i.v. dose) of the total 14C-activity administered. 2. Sulphamethazine accounted for 70-79% (oral dose) and 54-75% (i.v. dose) of the total 14C in milk samples collected from 0-48 h after dosing. N4-acetylsulphamethazine accounted for 1-2% (oral dose) and 1-4% (i.v. dose) of the 14C in milk. 3. The major 14C-labelled metabolite in the milk was isolated and identified as the N4-lactose conjugate of sulphamethazine, a unique type of metabolite not previously reported. This metabolite accounted for 10-14% (oral dose) and 9-20% (i.v. dose) of the 14C-activity in the milk collected from 0-48 h after dosing with 14C-sulphamethazine. 4. N4-lactose conjugates of sulphapyridine, sulphamerazine, sulphathiazole, sulphadimethoxine and sulphaquinoxaline were present in the milk from cows orally dosed with these five sulphonamide drugs.

Metabolism of 1,3-di-(4-[N-(4,6-dimethyl-2-pyrimidinyl)sulphamoyl] phenyl)triazene (DDPSPT) in the rat.

1. Six hours after rats were orally dosed with 1,3-di-(4-[N-(4,6-dimethyl-2-pyrimidinyl)sulphamoyl][U-14C]phenyl) triazene (14C-DDPSPT), approx. 81% of the 14C remained in the gastrointestinal tract (gut) and less than 3% was excreted in the urine. 2. Six hours after dosing, more than half of the 14C in the gut was present as DDPSPT. 14C-Labelled metabolites in the gut included 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)-benzenesulphonamide (Sulmet), N4-glucosyl-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamide (N4-gluc-Sulmet), 4-acetamido-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamide (N4-acetyl-Sulmet), and [N-4,6-dimethyl-2-pyrimidinyl) benzenesulphonamide] (desamino-Sulmet). 3. 14C-Labelled compounds in the blood, liver and skeletal muscle included DDPSPT, Sulmet, N4-gluc-Sulmet, N4-acetyl-Sulmet and desamino-Sulmet. 4. There was little or no reaction of DDPSPT with cysteine, bovine serum albumin, AMP, GMP, or calf thymus deoxyribonucleic acid in vitro (pH 3, 5, 7 or 8).

Characterization of products formed by the reaction of 14C-sulphamethazinediazonium tetrafluoroborate with natural products.

1. When bovine serum albumin (BSA) was incubated with 4-[N-(4,6-dimethyl-2-pyrimidinyl)sulphonamido] [U-14C]benzenediazonium tetrafluoroborate (14C-SDTFB) in vitro approx. half of the 14C-activity was bound (14C-BSA). Cysteine, N-ethylmaleimide, p-chloromercuribenzoate and iodoacetamide inhibited the formation of 14C-BSA. 2. When SDTFB was reacted with cysteine four major products were formed. These were identified as 3-(4-[N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamido] diazothio)-2-aminopropionic acid (cys-SDAS), 3-(4-[4,6-dimethyl-2-pyrimidinyl) benzenesulphonamido]thio)-2-aminopropionic acid (cys-Sulmet), 4-hydroxy-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamide (hydroxy-Sulmet) and N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamide (desamino-Sulmet). Diazosulphides were also formed when SDTFB was incubated with thiophenol and glutathione. 3. The diazosulphides reacted with N,N-dimethylaniline (DMA) and 2-naphthol to yield diazo compounds in 22-29% yield; when 14C-BSA was reacted with DMA under the same conditions, a diazo compound was formed-but only in 2% yield. 4. Cys-SDAS when incubated overnight (approx. 16 h) in aqueous solutions (pH 3, 5 and 8) decomposed to yield desamino-Sulmet (30-39%), hydroxy-Sulmet (13-21%), and other unidentified soluble products (24-36%); when 14C-BSA was incubated under the same conditions only 3-4% of the 14C became dissociated from BSA and only a trace amount of desamino-Sulmet was formed. 5. When 14C-SDTFB was incubated with calf thymus DNA at pH3, some of the 14C became associated with the DNA (14C-DNA). However, most of the 14C became dissociated from 14C-DNA when the latter was incubated overnight in aqueous solutions; a minor dissociation product was identified as 14C-desamino-Sulmet.

Diazomethane derivatization of sulfamethazine: formation of isomeric products.

Reaction of 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide (sulfamethazine) with diazomethane yields not only 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)-N-methylbenzenesulfonamide but also 2-(4-aminobenzenesulfonimido)-1,4,6-trimethyl-1,2-dihydro-pyrimidi ne. Yields of the latter compound are highly variable and the compound does not show a response to gas chromatography. Thus, results of gas chromatographic determinations of residues of some sulfa drugs in edible meat tissues may be erroneous when diazomethane derivatization is used.

The metabolism and deamination of [14C]sulphamethazine in a germ-free pig: the influence of nitrate and nitrite.

Twenty-four hours after feeding nitrite in combination with [14C]sulphamethazine to a germ-free and a conventional control pig the level of [14C]desaminosulphamethazine in tissues from both pigs was high, accounting for 11 to 30% of the total tissue 14C. When another germ-free pig was fed [14C]sulphamethazine in combination with nitrate, a trace amount of [14C]desaminosulphamethazine was found by gas chromatography-mass spectroscopy in the skeletal muscle but not in other tissues. When a control pig was fed [14C]sulphamethazine and nitrate, [14C]desaminosulphamethazine was found in all tissues examined. The results from this study show that feeding pigs nitrite together with sulphamethazine increases the amount of desaminosulphamethazine in the tissues. Most of the desaminosulphamethazine found in the tissues of the control pig fed nitrate was presumably the secondary effect of bacterial reduction of nitrate to nitrite.

Evidence for diazotization of 14C-sulfamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide] in swine. The effect of nitrite.

Dietary nitrite greatly enhanced the conversion of orally administered 14C-sulfamethazine (4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide; 14C-sulmet) to 14C-desaminosulfamethazine [N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide; 14C-DA-sulmet] in swine. The disposition of 14C orally administered to swine as 14C-sulfamethazinediazonium tetrafluoroborate (4-[N-(4,6-dimethyl-2-pyrimidinyl)sulfonamido] [U-14C]diazonium tetrafluoroborate) was very similar to the disposition of 14C given to swine as 14C-sulmet in combination with nitrite. These results and other information discussed in the text provide evidence that 14C-sulmet, in the presence of nitrite under the acid conditions in the gastrointestinal tract, was diazotized and that this diazonium intermediate was converted to 14C-DA-sulmet and other unidentified 14C-labeled products.

The disposition of N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide in the rat.

Rats given a single oral dose of N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide (14C-DAS) excreted 64.2% of the 14C in the urine and 22.4% in the feces within 96 hr. Compounds accounting for 86% of the 14C in the 0-24-hr urine were isolated by a variety of chromatographic techniques and identified by IR, NMR, and MS analysis. Approximately 4% of the 14C in the urine was the parent compound. The structures of 14C-metabolites in the urine indicated that 14C-DAS was metabolized by at least three pathways which included: 1) hydroxylation and glucuronic acid conjugation at the 4-position of the benzene ring; 2) hydroxylation, and sulfate ester and glucuronic acid conjugation at the 5-position on the heterocyclic ring; and 3) hydroxylation and glucuronic acid conjugation of one methyl group on the heterocyclic ring.

The effect of nitrite on 14C-sulphathiazole (4-amino-N-2-thiazolyl[U-14C]benzenesulphonamide) metabolism in the rat.

1. Rats given a meal containing 613 p.p.m. of 14C-sulphathiazole (4-amino-N-2-thiazolyl[14C]benzenesulphonamide) excreted less 14C-activity in urine and more 14C-activity in faeces as nitrite in the meal was increased (0, 10, 100 or 1000 p.p.m.). As nitrite in the meal was increased from 0 to 1000 p.p.m. the total 14C-residues in the gastrointestinal tract six hours after dosing increased, but decreased in other tissues. 2. High nitrite in the meal resulted in increased methanol insoluble 14C-activity in the gastrointestinal tract but had little or no effect on the methanol-insoluble activity in liver and blood. 3. Conversion of 14C-sulphathiazole to 14C-desaminosulphathiazole (N-2-thiazolyl[U-14C]benzenesulphonamide) in the rat was greatly increased by nitrite in the meal.

Formation of a diazonium cation intermediate in the metabolism of sulphamethazine to desaminosulphamethazine in the rat.

14C-Sulphamethazinediazonium tetrafluoroborate (14C-SDTFB) when orally administered to rats was converted primarily to 14C-labelled desaminosulphamethazine (desaminosulmet) and methanol-insoluble residues in the gastrointestinal tract (gut). 14C-labelled sulphamethazine (sulmet), N4-acetylsulmet, the N4-glucose conjugate of sulmet and other unidentified products were also observed in the tissues and urine of rats given 14C-SDTFB. 2. When 14C-sulmet, nitrite and dimethylaniline were simultaneously administered to a rat by the oral route, one of the 14C-labelled products formed in the stomach was isolated and identified as 4-dimethylaminophenyl [4-(N-4,6-dimethyl-2-pyrimidinyl)sulphamidophenyl] diazene, providing evidence that 14C-sulmet was diazotized in the stomach of the animal. 3. SDTFB was weakly mutagenic when evaluated by the Ames test. 4. The methanol-insoluble 14C-labelled residues in the gut of rats dosed orally with 14C-SDTFB and 14C-sulmet + nitrite were partially converted to 14C-labelled desaminosulmet, sulmet, N4-acetylsulmet and other unidentified products when fed to recipient rats.

Steady state kinetics of 14C-sulfamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide] metabolism in swine.

Swine were dosed orally with 14C-sulfamethazine [4-amino-N-(4, 6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide] for 3, 5, or 7 days (two 165-mg doses/day; 0.46 muCi/mg) and killed 8 hr after the last dose. The concentration of carbon-14 in the tissues increased by an average of 21% from day 3 to day 5 of dosing. However, there was no further increase from day 5 to day 7, indicating that a steady state level of carbon-14 in the tissues was attained by dosing on 5 consecutive days. Liver, kidney, skeletal muscle, blood, and adipose tissue from all animals were analyzed for 14C-labeled sulfamethazine, N4-acetylsulfamethazine, desaminosulfamethazine [N-(4, 6-dimethyl-2-pyrimidinyl)benzenesulfonamide], and the N4-glucose conjugate of sulfamethazine. The identity of these compounds (the hydrolysis product of N4-glucose conjugate) was confirmed by HLPC and gas-liquid chromatography/mass spectral analysis after methylation. The relative distribution of 14C-sulfamethazine and these metabolites varied somewhat among the tissues analyzed but did not vary within a tissue after different periods of dosing.