National Institute for Public Health and the Environment Annual CRL workshop 22 October Update on natural Hormone studies

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1 2008 Annual CRL workshop 22 October 2008 Update on natural Hormone studies

2 Natural hormone studies: update Possible approaches - C12/C13 ratio: can result in proof of abuse - Determination of intact esters traditionally limited to injection sites, significant advances due to hair analyses can result in proof of abuse - Determination of hormonal activity by the use of effect assays, inclusive marker assays. Primarily for screening - Determination of hormone profiles (multi analyte approach). Both precursors and metabolites (ratios). Extension with Phase II metabolites. Primarily for screening

3 Minisymposium 17 October 2007 Natural Hormones in Edible Tissues and Excreta James Scarth (UK, HFL) pattern analyses - Significantly more research therefore will be necessary, inclusive the development of new (multi analyte) methods, the collection of data from animals with known medication history and the setting of uni-/multivariate thresholds for ling the abuse of natural hormones Yoann Deceuninck (F, LABERCA) C12/C13 analyses - However, the analytical procedure is lengthy and complex and subsequently the costs are high. Moreover, with current state of the art techniques and equipment, its is not possible to achieve low µg/l sensitivity. The applicability therefore at the moment is limited. Statement to be updated November 2008

4 Minisymposium 17 October 2007 Natural Hormones in Edible Tissues and Excreta Robert Schilt (NL, TNO Ducares) - pattern analysis - Results until now indicate the presence of outlying values within homogenous groups of animals, indicating large physiological variability, analytical problems due to (extreme) large difference in concentration of individual compounds. Aldert Bergwerff (NL, UU-IRAS) effect assays - Based on the presence of a physiologically active compound (the effector), cells response, e.g. with the production of specific compounds. Such compounds can be biomarkers for the presence of e.g. hormones. Subsequently, analytical methods can be developed to detect such biomarkers in matrices like cattle plasma.

5 Minisymposium 17 October 2007 Natural Hormones in Edible Tissues and Excreta Jeroen Rijk (NL, RIKILT) - detection of prohormones - Studies focused on the possibility of bioactivation of prohormones on an analytical scale in order to make them detectable through effect based assays like the RIKILT androgen yeast Assay (RAA). Under laboratory conditions, it was demonstrated that DHEA can be activated and subsequently detected using the RAAs. In principle, this allows the use of the RAA for analyzing animal feed for pro(hormones).

6 Natural steroids meeting at Euroresidue VI. Sunday 18th May 2008 James Scarth HFL Ongoing activities: Development of multi residue methods for natural hormones, their precursors and metabolites Further development of methods for phase II-metabolites. Testing selected populations of samples. Further development and validation of methods for steroid esters in hair Further development of GC-C-IRMS

7 Research into natural steroid metabolism

8 Analytical method Natural Hormones in Serum and Urine

9 Steroid metabolism Cholesterol Pregnenolone 17α-Hydroxypregnenolone Dehydroepiandrosterone (DHEA) Progesterone 17α-Hydroxyprogesterone Androstenedione

10 Steroid metabolism Dehydroepiandrosterone (DHEA) 17β-Testosterone Androstenedione Estrone 17β-Estradiol

11 Testosterone phase I metabolites 17β-Testosterone 17α-Testosterone 4-androstenediol 17β-Dihydrotestosterone Androsterone Ethiocholanone (5β) 5α-Androstan -3β,17α-diol 5α-Androstan -3α,17β-diol 5α-Androstan -3β,17β-diol 5α-Androstan -3α, 17α-diol

12 Testosterone metabolism Dehydroepiandrosterone (DHEA) Androstenedione 17α-Testosterone 17β-Testosterone 17β-Dihydrotestosterone Androsterone Ethiocholanone 5α-Androstan -3β,17α-diol 5α-Androstan -3α,17β-diol

13 Collection of Reference values Bovine animals Sources are: Samples available at the CRL Reference blank samples Animal experiments Samples from other research Groups known history

14 CRL Reference Blank Samples Male Animals animals animal pregnant

15 Dehydroepiandrosterone (DHEA) Testosterone precursor Population Average values STDV Male ,0 40,0 39,0 38,0 37,0 36,0 35,0 34,0 33,0 32,0 31,0 pregnant Unknown population: male animals All values > A + 3 SD

16 Testosterone 17β-Testosterone Population Average values STDV 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 Male pregnant 3.3 < , Unknown population: male animals

17 5α-Androstan-3β,17α-diol Population Average values STDV Male Testosterone metabolite Femal pregnant ,0 90,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0? Unknown population: male animals

18 4-androstenediol Population Average values STDV Male Testosterone metabolite 10 7 pregnant No data

19 4-androstenedione Testosterone precursor Population Male Average values 5.4 STDV ,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0, pregnant Unknown population: male animals All values > A + 3 SD

20 17β-estradiol Estradiol Population Male Average values 0.2 STDV 0.6 2,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0? pregnant Unknown population: male animals

21 Estradiol metabolite Population Average values STDV 17α-estradiol Male ,0 250,0 200,0 150,0 100,0? pregnant ,0 0, Unknown population: male animals

22 Estrone Estradiol metabolite Population Average values STDV Male ,0 50, ,0 30,0 20,0 pregnant ,0 0, Unknown population: male animals One value exceeding A + 3 SD

23 Evaluation unknown population One animal with elevated value for 5α-Androstan-3β,17αdiol (Testosterone metabolite) and Estrone (Estradiol metabolite) All samples elevated value for 4-androstenedione and Dehydroepiandrosterone (DHEA) (precursor) No changes in testosterone levels Hair analyses proved treatment with testosterone esters!

24 Conclusions Analytical methodology is available for testing a range of natural hormones, their precursors and metabolites. Initial results confirm the high degree of variably and large datasets will be necessary for setting threshold values. Such threshold values as a rule will only be indicative Additional information could come from testing for Phase II metabolites Research activities for testing hormone-esters and GC-C- IRMS will continue

25 Continued work Analyses of led populations (treated and untreated) materials available? Further improvement of hair analyses (planned research study) Further study of phase II metabolites Continued evaluation of C12/C13 GC-C-irMS techniques