Sequencing CYP2D6 for the detection of poor-metabolizers in post-mortem blood samples with tramadol

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/Odesmethyltramadol), has been shown to be correlated with the poormetabolizer 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. We further state that this paper reports original work and is not under consideration for publication elsewhere. National Institute of Legal Medicine and Forensic SciencesCorresponding author: Suzana Fonseca Contacts:Email: sfonseca@dlinml.mj.pt

Heart insufficiency after combination of verapamil and metoprolol: A fatal case report and literature review

“…In this case, the TRA/ODT ratio was 15 indicating CYP2D6 PM genotype. The ratio TRA/ODT is not that specific in predicting CYP2D6 PM phenotype, but Fonseca et al found that a ratio between N‐desmethyltramadol (NDT) and ODT above seven was a more predictive ratio for CYP2D6 PM genotype . In this case, NDT/ODT = 56 gives a strong indication of our patient being CYP2D6 PM genotype.…”

“…The ratio between tramadol (TRA) and the metabolite O‐desmethyltramadol (ODT) can for living individuals be used to estimate an individual's CYP2D6 phenotype . In a postmortem setting, the ratio has been used to estimate an individual's CYP2D6 genotype . A TRA/ODT ratio above 15‐30 indicates CYP2D6 PM genotype.…”

Pharmacogenetics and Tramadol-Related Fatalities

“…The best correlation was obtained for M2/M1 ratio and PM. The M2/M1 ratio in PM is almost above 7 [48].…”

Intoxication by 3-MeO-PCP and O-desmethyltramadol: an unusual NPS mix

“…O-desmethyltramadol (O-DT) is the main metabolite of tramadol, a centrally acting opioid analgesic commonly prescribed for pain control in a therapeutic approach concerning postoperative, dental, cancer, neuropathic, and acute musculoskeletal clinical contexts, being responsible for the main opioid analgesic effect, approximately 300 times more affinity to μ-opioid receptors than the parent compound [12]. To the authors' knowledge, there are no published intoxication cases solely due to O-DT consumption.…”