Identification and detection of LH in urine
Luteinizing hormone (LH) is a naturally occurring hormone which is secreted by the pituitary gland. In males LH stimulates testosterone production by the testes. Recent research has proven that the monitoring of LH is a valuable tool for both the detection of anabolic steroid abuse; where suppression of LH is observed, and gonadotrophin releasing hormone abuse, where elevation of LH levels is observed. The ability to use LH as a marker for the detection of the abuse of other doping substances is limited by the currently available testing techniques. There are numerous commercially available immunoassays but these are only marketed for the testing of serum samples. The use of the available immunoassays for monitoring of LH in urine needs to be carefully cross validated for comparison to ensure suitably of the results for anti doping.
Furthermore to better understand why there are such differences in the concentrations detected by different immunoassays it is proposed to purify and sequence from urine both naturally present LH as well as from recombinant LH excretion studies. The proposed research will harmonize the measurement of LH in urine for anti doping as well as gain a better understanding of the measurand which will lead to better detection methodologies.
The Immulite and Delfia assay are able to detect LH in urine even after extended periods of storage at 40°C, 21°C, 4°C and -20°C. The results for the Delfia stability study reflect what was observed for the Immulite stability study, that LH is unstable at room temperature but although it is also unstable when frozen, quantifiable amounts are still able to be detected with the Delfia assay and the Immulite assay. Statistical analysis of 1000 athlete samples gave correlation factors for both the Immulite and the Delfia, enabling a more accurate comparison of results between the two assays. This will be particularly useful to the anti-doping community as different laboratories use only one or the other of the assays for detecting LH in urine. For anti-doping purposes urine is stored frozen which is where urinary endogenous LH is degrading. Sequencing of the beta subunit of LH revealed the C-terminal and N-terminal of the protein are still largely intact meaning that changes to the protein chain must be occurring within the protein backbone. The tertiary structure of LH may be allowing certain areas to more readily degrade due to turns and disulphide bond placements in the structure as well as the influence of post translational modifications such as the single N-linked glycan on the beta subunit.