Evaluating Sulfotransferases Metabolism In Vitro, Method Development and Application in Anti-doping Research

Abstract

Humans are constantly exposed to various xenobiotics, including environmental compounds like pollen, food, drugs, and pharmaceuticals. The metabolic processes occurring within the human body play a crucial role in regulating the activity and toxicity of these xenobiotics, facilitating their excretion before they accumulate to toxic levels. The metabolic pathway can be divided into two phases: phase I (functionalization) and phase II (conjugation) metabolism. Phase II metabolism involves two main pathways, glucuronidation and sulfonation, catalyzed by glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) respectively. Despite its significant role in both drug and doping compound metabolism, SULT catalyzed metabolism has not received sufficient attention over the years. This study focuses on the evaluation of the enzymatic functionality of human SULTs based on a recombinant fission yeast expression system, specifically in relation to doping compounds. The research aims to shed light on the role of human SULTs in drug metabolism and enhance our understanding of the significance of this pathway in regulating the metabolism of xenobiotics. The misuse of doping substances and methods is not only unethical and against the principles of fair competition but also poses significant health risks to athletes. Anti-doping testing is typically performed through the direct detection of administrated doping substances or their metabolites. Since numerous doping compounds undergo extensive metabolism after administration and remain nearly undetectable as parental compounds, their metabolites play an essential role in extending the detection window and are often considered preferred analytes. Detecting sulfate metabolites, particularly sulfate steroids (for their thermal instability), requires enzymatic or chemical hydrolysis of sulfates [3] as a pre-treatment. These processes can result in the degradation of certain analytes or the generation of undesired by-products, thus, additional purification steps prior to instrumental detection are necessary to avoid the by-product effects on analyte detection. Therefore, direct analysis of sulfate metabolites from urine excretion is desirable in anti-doping research. Despite the benefits of direct detection of sulfate metabolites, a significant challenge arises from the lack of sulfo-conjugates as standard reference compounds. Chemical synthesis of sulfate metabolites is considered efficient for compounds with only one potential conjugation site. By contrast, accurately synthesizing from compounds with multiple conjugation sites becomes problematic due to the potential side products, which may share a similar structure with the desired product and pose challenges in their separation. Thanks to the regioselectivity of enzymes, biosynthesis allows the synthesis of specific sulfate metabolites for compounds with multiple conjugation sites. In this work, I aimed to investigate a potential biosynthetic method as a viable alternative to the chemical synthesis of sulfate metabolites, mainly focusing on sulfo-conjugates of doping substances. The new method enhances the performance of SULT metabolite identification in doping control analysis and it also presents novel perspectives and valuable contributions for future research in this field.