Metandienone

In comparison to well-known drug-metabolizing organs such as the liver, the metabolic capacity of human skin is still not well elucidated despite the widespread use of topical drug application. To gain a comprehensive insight into anabolic steroid metabolism in the skin, six structurally related anabolic androgenic steroids, testosterone, metandienone, methyltestosterone, clostebol, dehydrochloromethyltestosterone, and methylclostebol, were applied to human keratinocytes and fibroblasts derived from the juvenile foreskin. Phase I metabolites obtained from incubation media were analyzed by gas chromatography-mass spectrometry. The 5α-reductase activity was predominant in the metabolic pathways as supported by the detection of 5α-reduced metabolites after incubation of testosterone, methyltestosterone, clostebol, and methylclostebol. Additionally, the stereochemistry structures of fully reduced metabolites (4α,5α-isomers) of clostebol and methylclostebol were newly confirmed in this study by the help of inhouse synthesized reference materials. The results provide insights into the steroid metabolism in human skin cells with respect to the characteristics of the chemical structures.

In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between 17α-Methyltestosterone-3-Carboxymethyloxime and Horseradish peroxidase label through N-hydroxysuccinimide mediated carbodiimide reaction method. The immunogen 17α-Methyltestosterone-3-Carboxymethyloxime-Bovine serum albumin used to generate the antibody was also prepared by the N-hydroxysuccinimide mediated carbodiimide reaction without using any spacer. We have studied the impact of bridge/aromatic spacers on functional parameters i.e. sensitivity, affinity and ED₅₀ of the bridge heterologous assay and compared it with homologous assay. The five combinations of bridge heterologous assay using 17α-Methyl testosterone-3-CMO-BSA antiserum and 17α-MT-3-CMO-4,4'-Diaminodiphenyl sulphide-HRP, 17α MT-3-CMO-4,4'-Oxydianiline-HRP, 17α-MT-3-CMO-Benzidine-HRP, 17α- MT-3-CMO-p-Phenylenediamine-HRP and 17α-MT-3-CMO-Dapson-HRP enzyme conjugates were evaluated. Out of these five combinations, the combination 17α-MT-3-CMO-BSA with 17α-MT-3-CMO-Benzidine-HRP showed the best results. Sensitivity, affinity and ED₅₀ were improved and found to be 0.02 ng/mL, 0.086 × 10^-8 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED₅₀ were 0.11 ng/mL, 0.02 × 10^-8 L/mol and 5.78 ng/mL respectively. The cross-reactivity for this bridge heterologous assay combination was seen with only 4 steroids (6-hydrotestosterone- 6%, Testosterone-5.14%, Danazol-0.9% and Nandrolone-0.85%) instead of eight steroids (6-hydrotestosterone-43.75%, Testosterone-38.3%, Danazol-25.14%, Androstenediol-19.16%, Nandrolone-19%, Metandienone-5%, Androstenedione-3.52%, and 17α dimethyltestosterone-2%) as in homologous assay out of 59 structurally related steroids. Thus, the results of this study conclude that the incorporation of aromatic spacer (bridge) in enzyme conjugate has a crucial role in improving sensitivity, specificity, ED₅₀ and affinity of the developed assay. The assay was then studied for parameters such as recovery (97.4%-108.6%), precision (Inter and Intra-assay coefficient of variation <10%), correlation coefficient (R² = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.