Abstract
Tramadol concentrations and analgesic effect are dependent on the CYP2D6 enzymatic activity. It is well known that some genetic polymorphisms are responsible for the variability in the expression of this enzyme and in the individual drug response. The detection of allelic variants described as non-functional can be useful to explain some circumstances of death in the study of post-mortem cases with tramadol. A Sanger sequencing methodology was developed for the detection of genetic variants that cause absent or reduced CYP2D6 activity, such as *3, *4, *6, *8, *10 and *12 alleles. This methodology, as well as the GC/MS method for the detection and quantification of tramadol and its main metabolites in blood samples was fully validated in accordance with international guidelines. Both methodologies were successfully applied to 100 post-mortem blood samples and the relation between toxicological and genetic results evaluated. Tramadol metabolism, expressed as its metabolites concentration ratio (N-desmethyltramadol/O-desmethyltramadol), has been shown to be correlated with the poor-metabolizer phenotype based on genetic characterization. It was also demonstrated the importance of enzyme inhibitors identification in toxicological analysis. According to our knowledge, this is the first study where a CYP2D6 sequencing methodology is validated and applied to post-mortem samples, in Portugal. The developed methodology allows the data collection of post-mortem cases, which is of primordial importance to enhance the application of these genetic tools to forensic toxicology and pathology.
Highlights
IntroductionTramadol is administrated in a racemic mixture and undergoes extensive phase I and II metabolization to 23 metabolites, mostly excreted in the urine
Tramadol is a centrally acting opioid analgesic commonly prescribed for treatment of postoperative, dental, cancer, neuropathic and acute musculoskeletal pain control, with high clinical efficacy, low incidence of adverse effects and low abuse potential.Tramadol is administrated in a racemic mixture and undergoes extensive phase I and II metabolization to 23 metabolites, mostly excreted in the urine
Post-mortem blood samples were studied searching for CYP2D6 genetic variants responsible for the enzyme inactivation, and the results obtained were compared with the concentration of tramadol and its main metabolites: NDT and ODT
Summary
Tramadol is administrated in a racemic mixture and undergoes extensive phase I and II metabolization to 23 metabolites, mostly excreted in the urine. The main metabolites resulting from the phase I metabolization are O-desmethyltramadol (ODT), catalyzed by CYP2D6 enzyme, and N-desmethyltramadol (NDT), catalyzed by CYP3A4 and CYP2B6 enzymes. Tramadol acts as a norepinephrine and serotonergic re-uptake inhibitor, possesses low affinity for μ opioid receptors and no affinity for δ or κ opioid receptors. The main opioid analgesic effect is attributed to ODT because it has approximately 300 times more affinity to -opioid receptors than the parent compound [1,2,3]. Post-mortem concentrations of tramadol are difficult to compare with reference values of therapeutic and toxic levels. Genotyping can be a useful tool to post-mortem toxicology to explain some unexpected concentrations of tramadol and parent/metabolite ratios
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