Predicting the likelihood of developing boar taint: early physical indicators in entire male pigs.

Three potential early-age predictors of which boars are likely to develop boar taint (testes volume, skin lesions and dirtiness) were measured on 102 boars every fortnight from 10 weeks of age until slaughter. These predictors were correlated with the level of boar taint according to the hot iron method and the concentrations of skatole and androstenone as determined by chemical analysis. The chance of no/low boar taint according to the hot iron method decreased with higher testes volume (weeks 22 and 24) and increased with skin lesion score (weeks 12, 16 and 18). For the concentrations of androstenone and skatole, the strongest correlation was found with testes volume in week 12. Skin lesions in week 16 were negatively correlated with skatole levels. Dirtiness was negatively correlated with skatole concentrations (week 18) but positively correlated with androstenone concentrations (weeks 20 and 22). Testes volume has the greatest potential for predicting the likelihood of developing boar taint.

Development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method for quantifying thyreostats in urine without derivatisation.

Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). For monitoring their illegal use, sensitive and specific analytical methods are required. In this study an UHPLC-MS/MS method was described for quantitative analysis of eight thyreostatic drugs in urine, this without a derivatisation step. The sample pretreatment involved a reduction step with dithiothreitol under denaturating conditions at 65 degrees C, followed by liquid-liquid extraction with ethyl acetate. This analytical procedure was subsequently validated according to the EU criteria (2002/657/EC Decision), resulting in decision limits and detection capabilities ranging between 1.1 and 5.5 microg L(-1) and 1.7 and 7.5 microg L(-1), respectively. The method obtained for all, xenobiotic thyreostats, a precision (relative standard deviation) lower than 15.5%, and the linearity ranged between 0.982 and 0.999. The performance characteristics fulfill not only the requirements of the EU regarding the provisional minimum required performance limit (100 microg L(-1)), but also the recommended concentration fixed at 10 microg L(-1) in urine set by the Community of Reference Laboratories. Future experiments applying this method should provide the answer to the alleged endogenous status of thiouracil.

Endogenous boldenone-formation in cattle: alternative invertebrate organisms to elucidate the enzymatic pathway and the potential role of edible fungi on cattle's feed.

Although beta-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of beta-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature.

Characterisation of steroids in wooden crates of veal calves by accelerated solvent extraction (ASE) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-QqQ-MS-MS).

Illegal steroid administration to enhance growth performance in veal calves has long been, and still is, a serious issue facing regulatory agencies. Over the last years, stating undisputable markers of illegal treatment has become complex because of the endogenous origin of several anabolic steroids. Knowledge on the origin of an analyte is therefore of paramount importance. The present study shows the presence of steroid analytes in wooden crates used for housing veal calves. For this purpose, an analytical procedure using accelerated solvent extraction (ASE(R)), solid-phase extraction (SPE) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-MS-MS) is developed for the characterisation of androstadienedione (ADD), boldenone (bBol), androstenedione (AED), beta-testosterone (bT), alpha-testosterone (aT), progesterone (P) and 17alpha-hydroxy-progesterone (OH-P) in wood samples. In samples of wooden crates used for housing veal calves, ADD, AED, aT and P could be identified. Using the standard addition approach concentrations of these analytes were determined ranging from 20 +/- 4 ppb to 32 +/- 4 ppb for ADD, from 19 +/- 5 ppb to 44 +/- 17 ppb for AED, from 11 +/- 6 ppb to 30 +/- 2 ppb for aT and from 14 +/- 1 ppb to 42 +/- 27 ppb for P, depending on the sample type. As exposure of veal calves to steroid hormones in their housing facilities might complicate decision-making on illegal hormone administration, inequitable slaughter of animals remains possible. Therefore, complete prohibition of wooden calf accommodation should be considered.

Determination of bixin and norbixin in meat using liquid chromatography and photodiode array detection.

The development of an analytical method that enables routine analysis of annatto dye, specifically bixin and norbixin, in meat tissue is described. Liquid-solid extraction was carried out using acetonitrile. Analysis was by HPLC with photodiode array detection using two fixed wavelengths (458 and 486 nm). The possibilities of ion trap mass spectrometry (MS) were also assessed. Method performance characteristics, according to Commission Decision 2002/657/EC, were determined, with recoveries between 99 and 102% and calibration curves being linear in the 0.5-10 mg kg(-1) range. The limit of quantification was 0.5 mg kg(-1).

Residue analysis: Future trends from a historical perspective.

A residue is a trace (microg kg(-1), ng kg(-1)) of a substance, present in a matrix. Residue analysis is a relatively young discipline and a very broad area, including banned (A) substances as well as registered veterinary medicinal products (B substances). The objective of this manuscript is to review future trends in the analysis of residues of veterinary drugs in meat producing animals out of historical perspectives. The analysis of residues in meat producing animals has known a tremendous evolution during the past 35-40 years. In the future, it can be foreseen that this evolution will proceed in the direction of the use of more and more sophisticated and expensive machines. These apparatus, and the necessary human resources for their use, will only be affordable for laboratories with sufficient financial resources and having guarantee for a sufficient throughput of samples.

Analysis of thyreostats: a history of 35 years.

Thyreostatic drugs (TS), illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). This paper reviews the trends in the analytical approaches for the determination of TS drugs in biological matrices. After a brief introduction on the different groups of compounds with a thyreostatic action, the most relevant legislation regarding the residue control of these compounds is presented. An overview of the analytical possibilities for the determination of TS in animal matrices, covering sample extraction, purification, separation techniques and detection methods is provided. Additionally, a brief outline of animal experiments is described that illustrates the excretion and distribution profiles of TS residues. Finally, the novel developments in TS analysis are highlighted. Also the possible semi-endogenous status of thiouracil is discussed.

Absence of an effect of dietary fibre or clinoptilolite on boar taint in entire male pigs fed practical diets.

This study aimed to evaluate the possibility of reducing boar taint in boars (Piétrain×Hybrid) by addition of different feed ingredients (raw potato starch (RPS) 10%, raw potato starch 10%+wheat bran 5% (RPS+WB), lupins 10%, inulin 5%, clinoptilolite 1%) to a standard diet over a period of 4-6 weeks before slaughter. Control boars (CBOAR) as well as barrows were fed the standard diet. Efficacy of the different feed ingredients was evaluated by different boar taint detection methods: hot iron method, consumer panel, expert panel and laboratory analysis. According to all detection methods, clear differences were noticeable between boars and barrows. No differences in boar taint incidence were found between the boars on the different dietary treatments as assessed by consumers, experts, hot iron method or the concentration of skatole in fat. A significant effect on indole level was found, but no further differentiation could be made. The concentration of backfat androstenone was significantly higher for the inulin and control boar group compared to the lupin group. In conclusion, none of the feeding strategies tested in this study reduced boar taint in boars at the given percentages.

Novel analytical methods for the determination of steroid hormones in edible matrices.

This paper reviews recently published multi-residue chromatographic methods for the determination of steroid hormones in edible matrices. After a brief introduction on steroid hormones and their use in animal fattening, the most relevant EU legislation regarding the residue control of these substances is presented. An overview of multi-residue analytical methods, covering sample extraction and purification as well as chromatographic separation and different detection methods, being in use for the determination of steroid hormones (estrogens, gestagens and androgens), is provided to illustrate common trends and method variability. Emphasis was laid on edible matrices and more specifically on meat, liver, kidney, fat and milk. Additionally, the possibilities of novel analytical approaches are discussed. The review also covers specific attention on the determination of natural steroids. Finally, the analytical possibilities for phytosterols, naturally occurring steroid analogues of vegetable origin and a specific group of steroid hormones with a hemi-endogenous status are highlighted.

Development and validation of a method for simultaneous analysis of the boar taint compounds indole, skatole and androstenone in pig fat using liquid chromatography-multiple mass spectrometry.

Boar taint in entire male pigs remains a problem for fresh pork production. Since castration of pigs will be prohibited in the future on animal welfare reasons, attempts are made to detect boar taint pre and post mortem. Post mortem techniques focus on simultaneous quantification of the three boar taint substances by one simple and reliable method. In this study a liquid chromatographic multiple mass spectrometric (LC-MS(n)) method has been developed and validated for the simultaneous determination of indole (2,3-benzopyrrole, ID), skatole (3-methylindole, SK) and androstenone (5alpha-androst-16-en-3-one, AEON) in pig fat samples. Sample preparation was kept as short as possible, since a single extraction method for structurally different indols and steroids was seeked after. Analytes were extracted from the fat matrix by methanol and clean-up consisted of freezing the extract in liquid nitrogen followed by a filtration step. The analytes were chromatographically separated on a Symmetry C(18) column. Recoveries for ID, SK and AEON, as calculated from fortified fat samples using 2-methylindole (2-MID) as internal standard, were 96, 91 and 104%, respectively. However, matrix interferences were encountered determining the androstenone levels in fat. Linearity, determined in fat samples, was in the range of 50-1600 microg kg(-1) for the indolic compounds and 125-2000 microg kg(-1) for the steroid AEON. The correlation coefficients (R(2)) of the calibration curves were 0.998 for ID, 0.997 for SK and 0.810 for AEON.

Past, present and future of mass spectrometry in the analysis of residues of banned substances in meat-producing animals.

A residue is a trace (microg kg(-1), ng kg(-1)) of a substance, present in a matrix. Banned substances, such as growth promoters, which are abused in animal fattening and where this article is focused on, may be divided into four major groups: thyreostats, anabolics or anabolic steroids, corticosteroids and beta-agonists or repartitioning agents. The combination of chromatographic techniques with mass spectrometry (GC-MS(n), LC-MS(n), etc.) plays a key role in the production of specific results in residue analysis. In this review, the past, present and future of mass spectrometry in this area are discussed in the light of the impact of these substances on human health and the reliable production of analytical results, ready for challenge in a court.

Multi-analyte approach for the determination of ng L(-1) levels of steroid hormones in unidentified aqueous samples.

Since the 1970s, many analytical methods for the detection of illegal growth promoters, such as thyreostats, anabolics, beta-agonists and corticosteroids have been developed for a wide range of matrices of animal origin, including meat, fat, organ tissue, urine and faeces. The aim of this study was to develop an analytical method for the determination of ng L(-1) levels of estrogens, gestagens, androgens (EGAs) and corticosteroids in aqueous preparations (i.e. drinking water, drinking water supplements), commercially available on the 'black' market. For this, extraction was performed with Bakerbond C18 speedisk, a technique commonly used in environmental analysis. After fractionation, four fractions were collected using a methanol:water gradient program. Gas chromatography coupled to electron impact multiple mass spectrometry (GC-EI-MS2) screening for the EGAs was carried out on the derivatized extracts. For the detection of corticosteroids, gas chromatography coupled to negative chemical ionization mass spectrometry (GC-NCI-MS) was used after oxidation of the extracts. Confirmation was done by liquid chromatography coupled to electrospray ionization multiple mass spectrometry (LC-ESI-MS2). The combined use of GC and LC coupled to MS enabled the identification and quantification of anabolics and corticosteroids at the low ng L(-1) level. This study demonstrated the occurrence of both androgens and corticosteroids in different commercial aqueous samples.

Phytosterols and anabolic agents versus designer drugs.

Cholesterol is a well-known component in fats of animal origin and it also is the precursor of natural hormones. Phytosterols appear in plants and only differ slightly in structure from cholesterol. An important difference however is the low absorption in the gut of phytosterols and their saturated derivatives, the phytostanols. As a result, there is time for all kind of reactions in faecal material inside and outside of the gut. Determination of the abuse of natural hormones may be based on gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Abuse of natural hormones changes the 13C/12C ratio of some metabolites during a relatively long time. The formation of (natural) hormones in the gut may interfere with this method. Designer drugs are mainly known from sports doping. In animal fattening, designer drugs may be used as well. Small changes in the structure of (natural) hormones may lead to a new group of substances asking for new strategies for their detection and the constatation of their abuse.

Mass spectrometric detection of and similarities between 1-androgens.

Regularly new anabolic steroids appear on the black market. In most cases these substances are marketed on websites or are confiscated during inspections. 1,(5alpha)-Androstene-17beta-ol-3-one, also known as 1-testosterone, is one of these substances presented to body-builders as a nutritional supplement or a pro-hormone. 1-Testosterone closely resembles the natural hormone testosterone except for a 1,2-double bound instead of a 4,5-double bound. 1-Androstene-3beta,17beta-diol is transformed into 1-testosterone after oral administration. 1-Testosterone, 1-androstene-3beta,17beta-diol and some other related 'new' anabolic steroids were studied with gas chromatography coupled to mass spectrometry (GC-MS) and Liquid chromatography coupled to tandem mass spectrometry (LC-MS2) methods. Similarities in spectra to known analytes, which may lead to pitfalls in the interpretation of the derivatised analytes, are discussed.

Formation of boldenone and boldenone-analogues by maggots of Lucilia sericata.

Current evidence suggests that neo formation of the anabolic steroid boldenone (androsta-1,4-diene-17-ol-3-one) occurs in calves' faecal material, making it difficult to distinguish between illegally administered boldenone and its potential endogenous presence. This strengthens the urgent need to elucidate the pathway leading to boldenone formation. In our laboratory, the invertebrate Neomysis integer (Crustacea, Mysidacea) was used since 2004 as an alternative model for the partial replacement of vertebrate animals in metabolisation studies with illegal growth promotors and veterinary drugs, e.g. boldenone. The present study evaluates the metabolic capacity of other invertebrates, the brine shrimp Artemia franciscana and maggots of the greenbottle fly Lucilia sericata. The first results indicate that maggots of L. sericata are able to convert phytosterols and -stanols, nowadays in substantial amounts added to animal feed, into androsta-1,4-diene-3,17-dione (ADD), the precursor of boldenone, at a yield of 0.10-0.14% (p<0.001, significance compared to endogenous excretion of maggots) but not to boldenone itself. Furthermore, beta-testosterone, an endogenous hormone, was transformed into androst-4-ene-3,17-dione (AED), ADD and beta-boldenone at a significant (p<0.001, significance compared to endogenous excretion of maggots) yield of circa 13%, 0.80% and 2.2%, respectively. In future studies these results are of value to further evaluate the use of maggots of L. sericata as an invertebrate model in metabolisation studies.

Testosterone metabolism in Neomysis integer following exposure to benzo(a)pyrene.

Cytochromes P450 (CYPs) are important enzymes involved in the regulation of hormone synthesis and in the detoxification and/or activation of xenobiotics. CYPs are found in virtually all organisms, from archae, and eubacteria to eukaryota. A number of endocrine disruptors are suspected of exerting their effects through disruption of normal CYP function. Consequently, alterations in steroid hormone metabolism through changes in CYP could provide an important tool to evaluate potential effects of endocrine disruptors. The aim of this study was to investigate the potential effects of the known CYP modulator, benzo(a)pyrene (BaP), on the testosterone metabolism in the invertebrate Neomysis integer (Crustacea; Mysidacea). N. integer were exposed for 96 h to 0.43, 2.39, 28.83, 339.00 and 1,682.86 microg BaP L(-1) and a solvent control, and subsequently their ability to metabolize testosterone was assessed. Identification and quantification of the produced phase I and phase II testosterone metabolites was performed using liquid chromatography coupled with multiple mass spectrometry (LC-MS2). Significant changes were observed in the overall ability of N. integer to metabolize testosterone when exposed to 2.39, 28.83, 339.00 and 1,682.86 microg BaP L(-1) as compared to the control animals.

The in vitro permeability of yeast peroxisomal membranes is caused by a 31 kDa integral membrane protein.

A major 31 kDa integral peroxisomal membrane protein (PMP31) of Hansenula polymorpha was purified to homogeneity from isolated peroxisomal membranes by FPLC after solubilization by Triton X-100. Biochemical analysis indicated that this protein, which showed cross-reactivity with antibodies against the 31 kDa porin of the mitochondrial outer membrane of Saccharomyces cerevisiae, had pore-forming properties. Firstly, proteoliposomes composed of asolectin and purified PMP31 showed selective permeability, determined as the [14C]sucrose/[3H]dextran leakage ratios. Furthermore, the generation of a delta psi by potassium diffusion gradients was negatively affected by the presence of PMP31 in asolectin liposomes. A similar effect was observed in proteoliposomes containing purified cytochrome c oxidase as a delta psi generating system. Control experiments confirmed that the observed leakage is significant and introduced by the incorporation of PMP31 protein. Selective sucrose leakage was abolished in samples pretreated with glutaraldehyde; an identical effect of glutaraldehyde was, however, not observed for the membrane potential measurements.

Presence of small GTP-binding proteins in the peroxisomal membrane.

Highly purified peroxisomal membranes stripped from their peripheral membrane proteins and only minimally contaminated with other membranes, contained three GTP-binding proteins of 29, 27 and 25 kDa, respectively. Bound radioactive GTP was displaced by unlabelled GTP, GTP analogs and GDP but not by GMP or other nucleotides. GTP binding was markedly decreased by trypsin treatment of intact purified peroxisomes; it increased 2-3-fold after pretreatment of the animals with a peroxisome proliferator. We conclude that the peroxisomal membrane contains small GTP-binding proteins that are exposed to the cytosol and that are firmly anchored in the membrane. We speculate that these proteins are involved in peroxisome multiplication by fission or budding during peroxisome biogenesis and proliferation.

Permeability properties of peroxisomal membranes from yeasts.

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH1 complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Single-channel analysis of a large conductance channel in peroxisomes from rat liver.

Native membranes and Triton X-100 solubilized integral membrane proteins of peroxisomes from rat liver were reconstituted in liposomes. With the patch clamp technique, a channel was detected with a conductance of 420 +/- 30 pS and a PK/PC1 of about 3. The channel in native membrane fractions was weakly voltage dependent, residing most of the time in an open state with the possibility to shift to different substates. Solubilization changed the kinetic properties. The channel became strongly voltage dependent and closed at voltages negative to -20 mV. The estimated diameter of the channel is about 1.7 nm and might explain, at least partially, the permeability properties of the peroxisomal membrane.