Gamma-tocotrienol (GT3) is one of the constituents of vitamin E that demonstrated significant radioprotective efficacy in murine and nonhuman primate (NHP) models. Considering the antioxidant activity of GT3 and its role in terminating lipid peroxidation, we hypothesize that mechanism of radioprotective effect of GT3 may involve the inhibition of irradiation-induced ferroptosis – a form of regulated cell death characterized by excessive, iron-dependent, peroxidation of lipids in cellular membranes. To test this hypothesis, the metabolomic and proteomic data from serum samples of GT3- or vehicle-treated NHPs exposed to 12 Gy (partial- or total-body) radiation were analyzed with focus on lipid peroxidation markers and proteins involved in iron metabolism. Four secondary lipid peroxidation products were identified, including 4-oxo-2-nonenal (4-ONE), 4-hydroperoxy-2-nonenal (4-HPNE), 3,4-epoxynonanal (3,4-ENA), and trans-4,5-epoxy-(2E)-decenal (4,5-EDE). In vehicle-treated animals, their concentrations increased significantly as soon as 4 h after irradiation and then gradually declined. GT3 treatment mitigated this radiation-induced increase. In addition to lipid peroxidation products, similar patterns of change were observed for several polyunsaturated, monounsaturated, and saturated fatty acids as well as amino acids such as lysine and its derivatives. Taken together, these metabolomic changes suggest that irradiation induces cellular membrane damage through enhanced lipid peroxidation, while GT3 exerts a protective effect against this process. In addition, GT3 increased serum levels of haptoglobin and hemopexin—two plasma scavenger proteins that play complementary protective roles in iron and heme homeostasis. Although the present study does not conclusively demonstrate that GT3 mediates radioprotection via inhibition of ferroptosis, the data suggest that GT3 limits membrane damage and reduces susceptibility to ferroptosis by enhancing iron and heme scavenging. Further investigation into the interaction between GT3 and key components of ferroptosis following exposure to ionizing radiation is therefore warranted.

Serum was separated from whole blood using serum separator tubes. After allowing blood to clot for 30 min, samples were centrifuged to isolate the serum, which was then placed into a new tube and stored at -80 °C until analysis. Metabolomic profiling was performed using UPLC-QTOF-MS. Metabolites were extracted from serum using a solvent mixture composed of methanol, isopropanol, water, and internal standards, followed by protein precipitation with acetonitrile. Centrifugation was performed to collect supernatants for analysis. Samples were then run on a UPLC system coupled to a Xevo G2 QTOF mass spectrometer using either a BEH or CSH C18 column for metabolomics or lipidomics, respectively. Mass spectrometry was performed in both positive and negative ionization modes, with regular injections of pooled quality control samples to monitor data consistency and instrument performance.