Prevalence of nandrolone preparations with endogenous carbon isotope ratios in Australia.

Nandrolone and its prohormones, including 19-norandrost-4-ene-3,17-dione and 19-norandrost-4-ene-3ß,17ß-diol, are anabolic steroids forbidden at all times in sports according to the World Anti-Doping Code Prohibited List and its metabolite 19-norandrosterone (19NA) is the preferred urinary target compound to identify their abuse. In recent years, an increasing number of 19NA isotope ratio mass spectrometry (IRMS) cases have arisen that, based on the initial testing procedure, were likely to result in an adverse analytical finding but were concluded negative after IRMS analysis. The current study was therefore set up to gain a better insight on the prevalence of nandrolone preparations with endogenous carbon isotope ratio values in Australia. Suitable workplace (non-athlete) urine samples that had previously been reported positive for 19NA were identified and analysed on IRMS. A total of 82% of the samples that were analysed were reported with enriched carbon isotope ratios of 19NA (i.e., 19NA greater than -26‰). This indicates that there is a high prevalence of nandrolone-containing anabolic androgenic steroid preparations in Australia that have 'endogenous' carbon isotope ratios which reduces the ability to identify exogenous nandrolone.

A multi-parametric approach to remove the influence of plasma volume on the athlete biological passport during a Union Cycliste Internationale cycling stage race.

Fluctuations in plasma volume (PV) present potential confounders within the concentration-based markers of the haematological athlete biological passport (ABP). Here, a multi-parametric approach involving a simple blood test is applied to the current ABP adaptive model in an attempt to remove the influence of PV expansion, induced by a cycling stage race. Blood samples were obtained from 29 professional cyclists (14 male, 15 female) before, during and after 4-5 consecutive days of racing. Whole blood was analysed in accordance with the World Anti-Doping Agency ABP guidelines for haemoglobin ([Hb]) concentration and platelets. Serum and plasma were analysed for transferrin, albumin, calcium, creatinine, total protein and low-density lipoprotein. PV variation (Z-scores) was estimated using a multi-parametric model (consisting of the biomarkers mentioned earlier) and compared against calculated variations in PV (measured via CO-rebreathing). Significant reductions in [Hb] and the OFF-score were observed in female cyclists after 3 and 4 days of racing, with accompanying increases in PV, which returned to baseline values 4 days post competition. Similarly, a significant increase in PV was observed in male cyclists after 3 and 5 days of racing. When individual estimations of PV variance were applied to the adaptive model, the upper and lower reference predictions for [Hb] and the OFF-score were refined such that all outliers consistent with racing-induced PV changes were removed. The PV model appears capable of reducing the influence of PV on concentration-dependent markers during competition. This is an important step towards the inclusion of the PV correction in the ABP haematological module.

Photochemical behavior and Na+,K+-ATPase sensitivity of voltage-sensitive styrylpyridinium fluorescent membrane probes.

RH421 is a widely used voltage-sensitive fluorescent membrane probe. Its exposure to continuous illumination with 577 nm light from an Hg lamp leads, however, to an increase in its steady-state fluorescence level when bound to lipid membranes. The increase occurs on the second time scale at typical light intensities and was found to be due to a single-photon excited-state isomerization. Modifications to the dye structure are, therefore, necessary to increase photochemical stability and allow wider application of such dyes in kinetic studies of ion-transporting membrane proteins. The related probe ANNINE 5, which has a rigid polycyclic structure, shows no observable photochemical reaction when bound to DMPC vesicles on irradiation with 436 nm light. The voltage sensitivity of ANNINE 5 was tested with the use of Na+,K+-ATPase membrane fragments. As long as ANNINE 5 is excited on the far red edge of its visible absorption band, it shows a similar sensitivity to RH421 in detecting charge-translocating reactions triggered by ATP phosphorylation. Unfortunately the wavelengths necessary for ANNINE 5 excitation are in a region where the Hg lamps routinely used in stopped-flow apparatus have no significant lines available for excitation.

Sensitizer-mediated photooxidation of histidine residues: evidence for the formation of reactive side-chain peroxides.

Exposure of proteins to visible light in the presence of a sensitizer results in the oxidation of Met, Trp, Tyr, Cys, and His side chains. These reactions are only partially understood, particularly with His. In this study, the oxidation of free His, His derivatives, and His-containing peptides has been examined using visible light and a range of sensitizers. It is shown that photooxidation gives rise to unstable peroxides, in a light-, illumination time-, and sensitizer-dependent manner. The yield of these materials is increased when reactions are carried out in solutions prepared with D2O, which prolongs the lifetime of 1O2, and decreased in the presence of the potent 1O2 scavenger azide, consistent with the involvement of this excited state. These peroxides have half-lives of hours, though the rate of decomposition is enhanced by elevated temperatures, reductants, and metal ions. Reducing metal ions catalyze the formation of radicals, which have been detected by EPR spin trapping. Structural analysis of His photo-products using NMR spectroscopy has provided evidence for the formation of oxygenated and cyclized compounds (e.g., 6a-hydroxy-2-oxo-octahydro-pyrollo[2,3-d]imidazole-5-carboxylic acid) and cross-linked materials. The latter materials may be partly responsible for the high yield of aggregated materials detected on photooxidation of His-containing proteins.