Stability of sulphadimidine during raw fermented sausage preparation.

The transformation of sulphadimidine (SDM) during raw fermented sausage preparation was studied to elucidate the SDM decrease found in an earlier study. The raw fermented sausages were prepared from batters containing 1 and 10 mg [14C]-SCM kg-1. The sausages and the brines were analysed using methods based on solid-phase extraction followed by HPLC combined with liquid scintillation counting. It can be concluded that the decrease in SDM level is mainly caused by (i) leaching into the brine (approx. 25%), (ii) transformation of SDM, possibly by reactions with components in the sausage or the brine, as the presence of five reaction products from SDM could be demonstrated, and (iii) formation of bound residues (approx. 20%).

Depletion of protein-bound furazolidone metabolites containing the 3-amino-2-oxazolidinone side-chain from liver, kidney and muscle tissues from pigs.

Ten 3-month-old pigs were treated with feed containing 300 mg furazolidone per kg for a period of 7 days, followed by withdrawal periods of 0, 1, 2, 3 or 4 weeks (two per group). The treatment resulted in the formation of protein-bound metabolites containing an intact 3-amino-2-oxazolidinone (AOZ) side-chain that could be chemically released and then detected in liver, kidney and rump muscle tissues even 4 weeks after dosing. In tissues from animals killed at the end of the medication period, 993, 600 and 124 ng of AOZ were released from 1 g of liver, kidney and muscle respectively. In the tissues of the animals killed after a further 4 weeks the corresponding levels were 41, 7 and 10 ng/g respectively. It may be concluded that long withdrawal periods prior to slaughter for human consumption are required for pigs treated with furazolidone, because of the long residence time of protein-bound AOZ and the possibility that it might be released from its protein-bound form in the stomach and subsequently be transformed into a hydrazine.

The use of pig hepatocytes to study the nature of protein-bound metabolites of furazolidone: a new analytical method for their detection.

The biotransformation of furazolidone, both in vivo and in vitro, results in the formation of so-called bound metabolites. Following the incubation of pig hepatocytes with [14C]furazolidone, a dose- and time-related increase in the formation of bound metabolites was observed. After withdrawal of the drug from the medium, levels decreased gradually to about 50% within 36 hr. Using a newly developed method, it was shown that in the case of cells and liver microsomes at least 70% of the bound residues still contained the 3-amino-2-oxazolidinone (AOZ) side chain of furazolidone. For liver samples of piglets that had been treated orally with the drug for 10 days, followed by withdrawal periods of 2 hr or 14 days, fractions of releasable AOZ were 23 and 14%, respectively, equivalent to levels of 3.8 and 0.3 micrograms furazolidone/g of tissue, respectively. The release of the AOZ side chain of furazolidone from at least part of the bound metabolites shows their drug-related nature and highlights the requirement for both adequate withdrawal periods and the need for appropriate control methods. The relative simplicity of the newly developed method may be helpful in further establishing and controlling adequate withdrawal periods.

The use of pig hepatocytes to study the biotransformation of beta-nortestosterone by pigs. Identification of glucuronidated 15 alpha-hydroxy-norandrostenedione as an important in vitro and in vivo metabolite.

Porcine hepatocytes were used to examine the biotransformation of beta-nortestosterone (NOR). Initially, the major metabolite of NOR was norandrostenedione (NA), which upon prolonged incubation was further transformed, primarily to the glucuronide of 15 alpha-hydroxy-norandrostenedione (15 alpha OH-NA). No differences were observed in this pattern between hepatocytes isolated from livers of sows or castrated male pigs. With increasing culture age, the rate of formation of NA did not change, contrary to a decrease in its further oxidation and glucuronidation. 15 alpha-OH-NA, primarily as its glucuronide, was identified in the urine of pigs injected with NOR. In addition, NA and the glucuronide of the parent compound were present in much smaller amounts.

Biotransformation of beta-nortestosterone by cultured porcine hepatocytes.

The metabolism of beta-nortestosterone by porcine hepatocytes was investigated. Initially beta-nortestosterone was rapidly oxidized to norandrostenedione, which was further transformed into a number of more hydrophilic compounds. It is assumed that most of these compounds were glucuronides, considering the effect of beta-glucuronidase treatment. The main product of enzymatic cleavage was investigated by gas chromatography-mass spectrometry but could not be identified until now.

Reversible interaction of a reactive intermediate derived from furazolidone with glutathione and protein.

Swine liver microsomes convert the nitrofuran furazolidone into N-(4-cyano-2-oxo-3-butenylidene)-3-amino-2-oxazolidone, a reactive open-chain acrylonitrile derivative. This derivative may be trapped with such thiol-group-containing agents as glutathione and mercaptoethanol. However, this reaction is reversible; e.g., adding an excess of mercaptoethanol to an aqueous solution (pH 7.4) of the glutathione conjugate results in conversion of 43% of this compound into the mercaptoethanol conjugate. In addition, when microsomal protein is added to the glutathione conjugate or the mercaptoethanol conjugate, 36 and 44%, respectively, become covalently bound to the protein. The amount of this covalently bound radioactivity decreases again on prolonged incubation at 37 degrees C (42% disappearance within 24 hr), suggesting that the acrylonitrile derivative also reacts reversibly with thiol groups of microsomal protein. Indeed an excess of mercaptoethanol could remove covalently bound radioactivity from microsomal protein resulting in the formation of the mercaptoethanol conjugate. The reversibility of the reaction is dependent on pH, as is demonstrated for the mercaptoethanol conjugate. Below pH 2 this conjugate is stable; optimal exchange to microsomal protein is found between pH 7 and 10. At very high pH (greater than 11) no binding to protein is found, although the conjugate disappears rapidly. The mercaptoethanol conjugate exhibits mutagenic activity in the Salmonella/microsome test indicating that the acrylonitrile derivative of furazolidone also interacts with DNA.

The elimination of furazolidone and its open-chain cyano-derivative from adult swine.

1. A sensitive method for the determination of 3-(4-cyano-2-oxobutylidene amino)-2-oxazolidone, the open-chain cyano-derivative of the veterinary drug furazolidone, in swine plasma and tissues is described. 2. After dosing adult swine orally with furazolidone (690 mg/animal per day) for 10 days no furazolidone was detected in liver, kidney and muscle (less than 2 ng/g). The half life of furazolidone as measured from the terminal phase of the plasma curves was 45 minutes. In urine, small amounts (less than 0.3% of total dose) of furazolidone were detected. 3. In contrast to other animals, 3-(4-cyano-2-oxobutylidene amino)-2-oxazolidone is a minor metabolite in swine with a plasma half life of 4 h. No cyano-derivative was detected in liver and kidney (less than 5 ng/g) 2 h after the last administration of furazolidone; 24 h after the last administration, the concentration in plasma was less than 2 ng/ml and in muscle less than 5/g. 4. The cyano-derivative was not mutagenic in the Salmonella/microsome test, with or without metabolic activation.