VETERINARSKI ARHIV 68 (3), 85-90, 1998

ISSN 0372-5480
Printed in Croatia

Indirect determination of thiouracils in slaughter cattle by measuring the mass of the thyroid gland

Darinka Z. Doganoc*, and Stanka Grebenc

Department of Food Hygiene and Bromatology, Veterinary Faculty,
University of Ljubljana, Ljubljana, Slovenia

* Contact address:
Doc. Dr. Darinka Z. Doganoc,
Department of Food Hygiene and Bromatology, Veterinary Faculty, University of Ljubljana, 1000 Ljubljana, Gerbiceva 60, Slovenia,
Phone: 386 61 1779 100, Fax: 386 61 332 243

DOGANOC, D. Z., S. GREBENC: Indirect determination of thiouracils in slaughter cattle by measuring the mass of the thyroid gland. Vet. arhiv 68, 85-90, 1998.

ABSTRACT

The use of thyreostatics (TS) in cattle breeding results in a considerable increase in live mass gain; therefore, its use is prohibited in most European countries. Thyreostatics, among which thiouracils (TU) are mostly used, lead to enlargement of the thyroid gland, which can be determined by measuring its mass. The illegal use of TS has been determined gravimetrically at the Veterinary Faculty, Institute for Food Hygiene and Bromatology, Ljubljana, since 1989. Up to the end of 1996 a total of 80 thyroids in pigs and 306 thyroids in cattle were examined. Only 12 samples of cattle thyroids exceeded a mass of 60 g. They were sent for confirmatory analysis, but the remainder was found to be negative. None of the 12 thyroids was histologically altered, and no residues of TU were detected. On the basis of our results it can be concluded that thiouracils were not used in cattle breeding, and all samples were TU-negative.

Key words: slaughter cattle, thyroid gland, tiouracils, thyreostatics

Introduction

Thyreostatics (TS) are substances that inhibit the formation of thyroid hormones. Their use in slaughter-cattle breeding leads to a mass increase in animals before slaughter. This is mainly due to increased water content and its retention in the gastrointestinal tract and in the muscular tissues (BATJOENS et al., 1993).

The thyroid gland (glandula thyroidea) is the largest endocrine gland, which, in mammals, is located at the windpipe, near the pharynx. Its two lobes (lobus sinister and lobus dexter) lie on either side of the thyroid cartilage and are connected by a bridge (isthmus).

The size and mass of the thyroid vary considerably, regardless of the species, and also individually with regard to the breed, age and sex, together with climatic conditions and season.

The thiouracils are different derivatives of pyrimidine and are best researched of all thyreostatics. The following are used: 4-hydroxil-2-mercaptopyrimidin (thiouracil, TU), 4 (6) methyl-2-thiouracil (methylthiouracil, MTU), 4 (5,6) dimethyl-2-thiouracil (dimethylthiouracil, DMTU), 4 (6) propyl-2-thiouracil (propylthiouracil, PTU), 4 (6) phenyl-2-thiouracil (phenylthiouracil, PhTU), 2-mercapto-1-methylimidazole (tapazole, TAP). The most frequently used are: MTU, PTU, PhTU, and Tapazole.

Since 1970, thiouracils have mostly been used as thyreostatics. Thyreostatic properties also possess other substances, particularly s.c. inorganic growth promoters such as ammonium perclorat (BATJOENS et al., 1993) and beta-agonists (SCHILT et al., 1996).

As already mentioned, a false mass increase is observed in slaughter cattle after feeding thyreostatics. Considering the economic benefits of such a practise, it will be difficult to prevent their use. Because the use of TS is prohibited throughout Europe, its abuse must be under rigorous and constant control. Not only is such meat of smaller consumer value, but the presence of TS residues in meat could constitute a health hazard. The control should be aimed in the first place at prevention of feeding thyreostatics, while the examination of tissues and excrements should constitute only a supportive measure. However, in addition to thiouracils as the most widely abused substances, our attention must also focus on other chemicals that possess thyreostatic activity. For some of them, methods of determination will have to be developed, along with their activity in the organism and the levels of residues in tissues.

The presence of TS may be determined by examination of various tissues and body fluids in animals. In most cases, urine, plasma and the thyroid gland are analysed. For control of the use of these substances, urine and the thyroid are predominantly used, as well as meat should TS residues constitute a health hazard.

In this context, much research has taken place around the world. In the Netherlands (VOS et al., 1982) thyroid glands of adult beef cattle were weighed and analysed by thin-layer chromatography (TLC) for the presence of TU, MTU, PTU, PhTU and methimazole. Glands registering positive with regard to the presence of residues varied in mass between 68 and 245 g. Glands in which no residues were found had a mass of between 20 and 124 g. Taking an upper limit of 60 g as normal, 22 glands were positive. The results of the TLC method compared favourably with the gravimetric method. On the basis of these results the gravimetric method is proposed as a "screening" test for the detection of thyreostatics.

In 1986, studies were performed in Switzerland to detect the illegal use of TS in slaughter cattle by weighing the thyroid glands (VENZIN et al., 1986). Of 328 analysed glands, none was MTU- or TU-positive. Three of 275 glands exceeded a mass of 80 g.

In 1987 in Italy, three different methods to detect the illegal use of TS: histological, gravimetric and chemical two-dimensional HPTLC (PALLIOLA et al., 1987) were compared one with the other. A total of 68 thyroids of adult bovines at regular slaughter were analysed. Through chemical analysis it was established that the glands were TS negative, whether or not histological changes occurred, where the mass did not exceed 50 g. The mass of 5 thyroids exceeded 50 g and 3 were MTU-positive. It was established that the mass increase of the thyroid was somehow associated with the presence of TS residues. With a mass of 50 g as the upper limit, it was decided to control only those glands exceeding this mass by means of chemical analysis.

Materials and methods

The control of illegal use of TS in slaughter cattle began in Slovenia in 1989 within the framework of a regular, annual veterinary meat inspection service. Control of thyroid glands was introduced in our export slaughterhouses in compliance with EEC and U.S.A. regulations. The gravimetric method was used which, together with HPTLC and HPLC, is prescribed by the EEC.

Samples

In accordance with regulations laid down by the Veterinary Administration of the Republic of Slovenia, thyroid glands were removed at slaughter and brought to the laboratory of The Institute of Food Hygiene and Bromatology, where they were trimmed, mass measured and stored prior to analysis. Up to the end of 1995 a total of 80 pig glands and 246 cattle glands had been examined. Of that number, 59 cattle glands came from Murska Sobota, 13 from Maribor, 39 from Celje, 20 from Ljubljana, 13 from Nova Gorica, and 6 from Secovlje.

In 1996, sixty cattle thyroids were examined: 12 from Ljubljana, 12 from the Maribor region, 11 from Nova Gorica, 9 from Celje, 5 from Murska Sobota, 5 from the Capodistria region, 3 from Gorenjska, and 3 from the Gornja Radgona region (DOGANOC et al., 1997).

Methods

Residues of TS in thyroids were determined gravimetrically. In samples exceeding the upper limit of 60 g, histological tests were used. The confirmatory tests and chemical analyses were carried out on our behalf in the Chelab laboratories in Italy.

Results and discussion

During the period from 1989 to 1996, a total of 306 thyroid glands of cattle were examined. The majority of them (54.9%) had a mass less than 20 g. Twelve glands (3.9%) exceeded a mass of 60 g. In Table 1 the number of examined thyroid glands, as well as their distribution into classes with regard to gland mass in a particular year, are shown. Until the end of 1995, eleven glands (4.5%) exceeded a mass of 60 g. None of them was histologically altered. In 1996, the thyroid glands of 9 calves were examined (mean value of 12.7 g), 39 glands of young cattle up to 24 months of age (mean value of 21.4 g), 9 cows (mean value of 29.1 g) and 2 bulls over 2 years of age (mean value of 14.85 g). In that year only one sample of thyroid gland exceeded the upper limit of 60 g. This gland came from a two-year-old bull, raised in Slovenske Konjice. The sample (with a mass of 76 g) was sent for analysis to the Chelab laboratory, where it was established that it was MTU-, PhTU-, PTU- and TAP-negative, respectively, with each concentration being <0.001 mg/kg. Results of all 60 cattle thyroids examined in 1996 were negative.

Table 1. Number of examined thyroid glands of cattle in a particular year, and distribution into classes with respect to the mass of gland

* Values of the individual mass of thyroid glands were: 66.7 g; 135.9 g; 66.3 g; 89.8 g; 97 g; 64.7 g; 70,6 g; 104.7 g; 99.2 g; 68.0 g; 104.1 g; 76.0 g

The upper limit of the mass of the thyroid gland, fixed at 60 g in agreement with the Veterinary Administration of Slovenia on the basis of various data from the literature (VOS, 1982; VENZIN, 1986), has proved to be reasonable and will, therefore, be applied in the future. It was established that the gravimetric method is effective as a screening test (VOS, 1982) although a chemical method must be employed for thyroids with a markedly increased mass (HOOIJERNIK, 1987; MORETTI, 1988).

The results of our work showed that the thyroid glands of our cattle were negative with respect to TS residues. However, on the basis of our research it is not possible to conclude that there is no illegal use of TS in our country. In certain tissues, trace amounts of TS can be detected only for a short period of time after their use. Therefore, producers and importers of veterinary medicines should be controlled initially for the illegal use of TS. Such a method of control would be more effective and, more importantly, more economic, as the detection of small amounts of TS in meat is extremely difficult and prevents an effective control of the use of these substances.

References

BATJOENS, P., H. F. De BRABANDER, L. T'KINDT (1993): Ion chromatographic determination of percheorate in cattle urine. Analytica Chimica Acta 275, 335-340.

DOGANOC, D. Z., J. MARINSEK, K. ŠINIGOJ GACNIK, V. CERKVENIK, S. GREBENC (1997): Veterinarsko-sanitarni nadzor nad zivili v Sloveniji - 1996. Ostanki onesnazevalcev okolja in veterinarsko-medicinskih preparatov v zivilih zivalskega izvora. Ministrstvo za kmetijstvo, gozdarstvo in prehrano, Veterinarska fakulteta, Ljubljana, 104 p.

HOOIJERNIK, H., W. G. De RUIG (1987): Determination of thyreostatics in meat by revessed-phase chromatography with ultraviolet and electrochemical detection. J. Chromatogr. 394, 403-407.

MORETTI, G., M. AMICI, P. CAMMORETA, F. FRACOSSI (1988): Identification of thyreostatic residues of animal thyroid by high-performance thin-layer chromatography and fluorescence reaction detection. J. Chromatogr. 442, 459-463.

PALLIOLA, E., G. MORETTI, A. NANNI, M. AMICI, C. ROSSI (1987): Valutazine degli esami istologico, ponderale e chimico sulla tiroidi dei bovini quali elementi per il controllo del trattamento illegale con tireostatici. Ann. Ist. Super. Sanita. 23, 129-134.

SCHILT, R., E. O. von BENNEKOM, L. BRONWER, H. HOOIJERINK, P. STONTEN, W. HAASNOOT, M. J. GROOT, P. L. M. BERENDE, F. A. HUF (1996): Levels of beta-agonists in urine, faeces, liver and retina/chorid after treatment of male veal calves with a combination of beta-aginists-influence of co-medication with estradiol, methylthiouracil and dexamethasone. Conference EURO RESIDUE III. B 17 (last minute poster contribution).

VENZIN, J., J. BLUM, F. CRAMERI, F. EHRENSPERGER, H. LUTZ (1986): Thyreostatika - Missbrauch beim Rind: Bestimung der Thyroxin - (T4) - Konzentration und des Schilddrüsengewichtes und Nachweis von Methylthiouracil (MTU). Schweiz. Arch. Tierheilk. 128, 443-458.

VOS, J. G., R. V. STEPHANY, J. W. CASPERS, J. Th. G. VON LOON, J. W. H. METZLAR, H. B. M. OBERHAUS (1982): Weight increase of the thyroid gland as a tentative screening parameter of detect the illegal use of thyreostatic compounds in slaughter cattle. Vet. Quart. 4, 1-4.

Received: 29 January 1998
Accepted: 20 May 1998

DOGANOC, D. Z., S. GREBENC: Posredno utvrdivanje tiouracila u goveda vaganjem stitne zlijezde. Vet. arhiv 68, 85-90, 1998.

SAZETAK

Upotreba tireostatika (TS) u uzgoju stoke za klanje nerealno poveca masu zivotinja i zbog toga njihova upotreba nije dozvoljena u mnogim europskim zemljama. Tireostatici, medu kojima se tiouracili (TU) najviše koriste, uzrokuju povecanje stitne zlijezde, što mozemo ustanoviti vaganjem. Ilegalna upotreba TS u uzgoju stoke u Sloveniji kontrolirana je na Institutu za higijenu ziveznih namirnica na Veterinarskom fakultetu u Ljubljani. Od 1989. godine koristi se gravimetrijska metoda. Od 1989. do kraja godine 1996. pretrazeno je bilo 80 stitnih zlijezda svinja i 306 stitnih zlijezda goveda. Samo 12 zlijezda je preslo masu od 60 g. Ti uzorci su bili analizirani i kemijskim analizama. Uzorci nisu bili histološki promijenjeni i u njima nisu bili nadeni ostaci TU. Na osnovi tih rezultata utvrdili smo, da u uzgoju stoke TU nisu bili upotrijebljeni i uzorci nisu sadrzavali ostataka TU.

Kljucne rijeci: goveda za klanje, stitne zlijezde, tiouracili, tireostatici