Application of a novel UPLC-HRMS-based plasma metabolomics approach reveals differences between male and female mice following i.p. administration of trans-crocin-4.

Trans-crocin 4 (TC4) is an important carotenoid constituent of saffron showing remarkable activity against Alzheimer's disease due to its antioxidant and antiamyloidogenic properties. Metabolomics is an emerging scientific field that enhances biomarker discovery and reveals underlying biochemical mechanisms aiming towards the early subclinical diagnosis of diseases. So far, there have been scarce data demonstrating changes induced to mice plasma metabolome after TC4 administration. Thus, an untargeted UPLC-HRMS metabolomics approach has been employed to determine the alteration to the metabolic fingerprint after i.p administration of TC4 in male and female mice. Therefore, blood samples from 58 mice administered with TC4 including control animals were analyzed by UPLC-HRMS (Orbitrap Discovery XL). Statistical evaluation of the results was achieved by multivariate analysis (MVA) i.e. principal component analysis (PCA), projections to latent structures-discriminant analysis (PLS-DA) in order to discover the variables that contributed to the discrimination between treated and untreated groups whereas the variables contributing to the discrimination were identified using comparisons to online databases (e.g. Metlin) along with software manipulations e.g. adduct and fragment identification, covariance searching etc. It should be noted that due to the high variability imposed by various factors e.g. sex, dose and time points, the multilevel PLS-DA e.g. splitting variation to each individual component, has proven to be the only effective approach. By this methodology, the time sequence of metabolome changes due to the administration of TC4 has been made apparent. Furthermore, a sex-related effect on the metabolome has been proven to exist, denoting that the administration in both genders is indispensable in order to acquire safe conclusions as reliable metabolome pictures.