Development and validation of an enzyme-linked immunosorbent assay for the detection of circulating antibodies raised against growth hormone as a consequence of rbST treatment in cows.

The recombinant bovine growth hormone (rbST) is used to increase lactating performances of dairy cows. Administration of rbST is banned in the European Union; nevertheless, its use is probable. Until now, efficient analytical strategies to detect such practice are based on the direct detection by mass spectrometry of the presence of rbST in biological fluids, which suits for confirmatory purposes. Current screening strategies do not offer satisfactory performances; therefore, alternative screening strategies are required. The aim of the present work is to develop and validate an ELISA to measure the production of specific antibodies upon rbST in bovine sera. In this immunoassay, rbST is absorbed onto microtiter plate. After specific purification of the antibodies in serum, samples are analysed and the presence of antibodies anti-rbST is detected by Protein G peroxidase conjugate and 2-2'-azino di-ethyl benz-thiazoline-6-sulphonic acid (ABTS). The mean reproducibility of the OD (λ=405 nm) measurement was calculated with a CV of 13%. The intra- and inter-assay CVs ranged from 0.79% to 7.91% and from 2.69% to 20% respectively. The test presents cross-reaction with other growth hormones such as the recombinant equine (reST) and porcine (pST) (100% and 80% respectively). The specificity of the test toward rbST anabolic treatment was confirmed through the analysis of sera samples collected on animals administered with other anabolic compounds (steroids). The performances of the present anti-rbST ELISA proves its efficiency as a new screening tool to highlight illegal administration of rbST in cattle up to at least 3 weeks after treatment.

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle 1. Metabolite profiles of boldenone, boldenone esters and boldione in cattle urine.

Boldenone is an androgenic steroid that improves the growth and food conversion in food producing animals. In most countries worldwide, this anabolic steroid is forbidden for meat production. Until recently, the control of its illegal use was based either on 17beta-boldenone or 17alpha-boldenone (its main metabolite in cattle) identification in edible tissues, hair, faeces or urine. Recent observations and data tend to demonstrate the natural occurrence (but not ubiquitous) in cattle of these steroids, making the analytical strategy of the control more complicated. We investigated the metabolism of boldenone in cattle after intramuscular and oral treatment of boldenone, boldenone esters and boldione. The central objective was to elucidate the structures of the main metabolites (phase I and phase II) in urine, with main objective to be further in position to compare boldenone urinary profiles of treated and non-treated animals. Nine metabolites have been identified, only four were present whatever the treatment and the administered boldenone source. Nevertheless, all of them have been detected at least once in non-treated animals which did not permit us to use them as biomarkers of an illegal treatment. At last, but not at least, all metabolites were found mainly glucuro-conjugated, and rarely sulfo-conjugated, with the only exception of 17beta-boldenone. Current investigations are showing the absence of 17beta-boldenone sulfoconjugate in non-treated animals; that would permit to distinguish non-treated from treated animals with boldione, boldenone and boldenone esters.

Endogenous nandrolone metabolites in human urine. Two-year monitoring of male professional soccer players.

19-Norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) are the two main indicators used to prove the illegal use of nandrolone by humans. Recent studies showed that 19-NA and 19-NE can be endogenously produced in some individuals. The mediated cases observed over the last three years generated some questions about the appropriateness of the official International Olympic Committee cutoff level, which is 2 ng/mL of 19-NA in male urine samples. In the present study, professional soccer players belonging to the French First League were studied over a period of 19 months. In total, 385 urine samples were taken immediately before and after soccer competitions and were coupled with 200 blood samples for testosterone and LH determination. Results of the study showed that the mean values for 19-NA and 19-NE were 0.097 ng/mL and 0.033 ng/mL, respectively. For 19-NA, 70% of the samples proved to be below 0.1 ng/mL, whereas less than 20% were found to be between 0.1 and 0.2 ng/mL, and 7% were between 0.2 and 0.3 ng/mL. Only four urine samples were above 1.0 ng/mL; the maximal value was 1.79 ng/mL. For 19-NE, only one sample was above 1.0 ng/mL; the value was 1.42 ng/mL. Concentrations of these compounds after games were generally significantly higher than those before games.

Endogenous nandrolone metabolites in human urine: preliminary results to discriminate between endogenous and exogenous origin.

When administered to human subjects, nandrolone is metabolized into two main products, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Recent studies demonstrated the endogenous production of these compounds in man at concentrations very close to the threshold of the International Olympic Committee (IOC), i.e. 2 ng/ml. Because the possibility of reaching or exceeding this fateful limit is difficult to exclude, a complementary biochemical parameter is necessary for the differentiation of endogenous 19-NA and 19-NE production from residues resulting from nandrolone consumption. We measured the endogenous concentrations of 19-NA and 19-NE in 385 urine samples from professional football players, and we studied the phase II metabolite composition in individuals excreting the highest concentrations. The results showed that around 30% of endogenous 19-norandrosterone was sulfo-conjugated, whereas 100% of 19-norandrosterone was excreted conjugated to a glucuronic acid when nandrolone was administered. This significant qualitative difference appears to be a promising complementary criterion to more definitively conclude about an athlete's culpability, especially when nandrolone metabolites are found in the low ng/ml range.