Vitamin E, one of the essential micronutrients and a known antioxidant, includes two groups of closely related fat-soluble compounds, the tocopherols and tocotrienols, each with the four analogues, α, β, γ, and δ. These isoforms differ in the saturation of their side chains and both the number of methyl substituents and their positions in the chromanol ring [1, 2].
For the most part, early antioxidant studies focused on α-tocopherol, the predominant form of vitamin E existing in human and animal tissues. Based on epidemiologic and retrospective studies, supplementation with Vitamin E (mainly α-tocopherol) is considered to be a nutrition-based strategy to prevent diseases and to support healthy aging. However, a new study in mice showed that α-tocopherol can stimulate bone osteoclast fusion independent of its antioxidant activity, resulting in increased bone resorption . The results from that study raise a question of whether vitamin E (mainly α-tocopherol) taken as dietary supplement is suitable considering it may have a risk of osteoporosis.
On the other hand, tocotrienols have gained increasing scientific interest during recent years as they have been reported to possess certain biology activities that were not observed in tocopherols . This is especially true for γ-tocotrienol (GT3), which is abundant in palm oil and rice bran [5, 6], as it provides significant health benefits, including anticancer [7–10], anticholesterolemic [11, 12], antihypertensive , and antiatherosclerotic effects , as well as acting as a potent antioxidant [15, 16]. Moreover, GT3 is a particularly potent radio-prophylactic agent in vivo  and has favorable effects on bone . All of these studies have shown γ-tocotrienol health benefits to be significantly greater than those of α-tocopherol.
Despite the growing interest in GT3, there is a paucity of information about bioavailability and there is still much controversy about its absorption, retention, and metabolism. α-tocopherol is preferentially incorporated into very low-density lipoproteins and transported to various tissues by lipoprotein because of its high affinity for α-tocopherol transfer protein (α-TTP) . In contrast, the absorption of tocotrienols has been found to be low and incomplete by oral administration . The presence of a transfer protein that preferentially selects α-tocopherol seems to explain why all other forms of vitamin E have a lower biological activity compared with α-tocopherol. Therefore, oral administration is unsuitable for GT3 biological activity studies in vivo.
In the previous studies of radioprotection, we found that administration of a single-dose of GT3 with polyethylene glycol (PEG-400) emulsion by subcutaneous injection could ameliorate radiation injury, including radiation-induced vascular oxidative stress, gastrointestinal injury, protection of hematopoietic stem and progenitor cells depletion, and induction of cytokines production. These effects led to enhancement of hematopoietic recovery and improvement of 30-day survival after total body irradiation (TBI) [21–23]. These results suggested that administration of emulsified GT3 by subcutaneous injection has long-term biological effects; however, whether the biological effects are due to the increase of GT3 level in the early phase or the persistent functions as the accumulation in tissues is unknown. Therefore, it is necessary to investigate the biodistribution of emulsified GT3 and the duration of biological activity in vivo after a single-dose of GT3 administration.
Receptor activator of NF-κB ligand (RANKL) is a member of the tumor necrosis factor (TNF) family of cytokines and is expressed by stromal/osteoblast, hypertrophic chondrocytes, osteocytes and other cell types, including T and B lymphocytes . Binding of RANKL to its receptor RANK triggers the activation of multiple downstream signaling which regulates osteoclast formation, activation and survival in normal bone modeling and remodeling . Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL that blocks ligand binding to RANK, thereby preventing the signaling required for osteoclast differentiation and activation . The relative concentration of RANKL and OPG in bone is a major determinant of bone mass and the RANKL/RANK/OPG system is critical for skeletal health, disruption of this system leads to or causes numerous bone diseases . Previous studies have reported that γ-tocotrienol can inhibit osteoclast formation and improve normal bone structure [28, 29].
In this study, we determined the durative levels of GT3 in different tissues by high performance liquid chromatography (HPLC) with a fluorescence detector after administration of a single-dose of GT3 with polyethylene glycol (PEG-400) emulsion by subcutaneous injection. In addition, we also investigated the persistent biological activity of accumulated GT3 by examining RANKL and OPG gene expression in bone tissues at various time intervals following db-cAMP administration.