Calving out protein biomarkers for anabolic steroids

Cattle are such an important food source around the world that many countries permit the use of growth promoting steroid hormones to bulk up the animals and increase the yield of meat. The USA and Canada are two countries that follow this practice but the EU has banned the use of hormones in cattle due to health concerns. It has also banned the import of hormone-treated beef, leading to a longstanding trade dispute with North America.
Although the ban is in force throughout the EU, enforcing it can be tricky. Detection can be evaded if tests are carried out too late. The steroids have metabolised, performed their anabolic functions and are no longer detectable. Some steroids metabolise more quickly than others and disappear without trace.
Two common steroid hormones that are used to bulk up cattle are 1,4-androstadiene-17beta-ol-3-one (beta-BOL), known as boldenone, and its 3,17-dione analogue boldione. The metabolisms of these steroids are closely related. In calves, boldione is a direct precursor of beta-BOL and has also been identified as a precursor and metabolite of alpha-BOL, the inactive epimer of beta-BOL. The major epimer in cattle urine is alpha-BOL and this is currently used as a marker for steroid misuse.
The EU authorities have declared that the presence of alpha-BOL conjugates in cattle urine at levels greater than 2 ng/mL should be regarded as suspicious. Similarly, the presence of beta-BOL conjugates at any concentration in urine is also evidence of misuse, since the natural levels of that epimer are very low. To enforce these limits, LC/MS methods have been developed to detect boldione and both BOL epimers in cattle urine, as well as in faeces, feedstuffs and skin swabs.
However, Italian researchers have noted that there is no published method for the detection and measurement of these three compounds in bovine plasma, so they set about developing one. Elisabetta Cosulich from the University of Genoa, with colleagues from four other organisations, treated veal calves orally with a boldenone/boldione mixture based on a steroid cocktail seized from a farm by the Italian police. Both blood and urine were collected up to 36 hours after the administration of a single dose.
In the first instance, the team developed an LC/MS/MS method with atmospheric pressure chemical ionisation (APCI) to measure the three compounds in urine before and after deconjugation, and in plasma. Boldione was not detected at all in either fluid despite detection capabilities of 0.3 ng/mL. Both BOL epimers were detected initially in both fluids, but were undetectable in plasma 2 h after administration and in urine after 24 h.
For animal testing, these represent short intervals, so the team decided to see if a proteomics approach might be more rewarding. They reasoned that the administration of steroid hormones might change the production of one or more proteins in some way. So, the proteins in plasma collected before and after drug dosing were separated by two-dimensional gel electrophoresis and the protein spots were stained and their intensities compared by image analysis.
Over the 36 h of sampling, one protein spot stood out from the hundreds visualised. It was absent before drug administration but was visible in the first post-drug sample (2 h) and increased in intensity steadily over time up to 36 h. This protein was identified as an N-truncated form of apolipoprotein A1 (ApoA1).
Its post-dose variation was confirmed by Western immunoblotting experiments conducted with an antibody against ApoA1. Two ApoA1-related bands were observed before drug treatment but a third band corresponding to the N-truncated form appeared after treatment at the correct molecular mass value and its intensity increased with time.
The researchers conjectured on the role of N-terminal proteolysis of ApoA1 in the presence of anabolic steroids, suggesting that the steroids might induce conformational changes in the protein, exposing its N-terminus to proteolytic attack.
They recognise that far more extensive and rigorous testing must be carried out over a longer timescale before this truncated ApoA1 can be accepted as a biomarker for the misuse of boldenone/boldione in animal husbandry. Immunoblotting could provide a simpler way forward for these large-scale screening and dose-dependent tests.
If the effect is confirmed, then the appearance of ApoA1 in bovine serum will serve to signify illegal use of these hormones when the plasma or urinary levels of the steroids themselves have returned to normal levels.

source:spectroscopynow.com