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In nature, eight substances have been found to have vitamin E activity: α-, β-, γ- and δ-tocopherol; and α-, β-, γ- and δ-tocotrienol. Yet, of all papers on vitamin E listed in PubMed less than 1% relate to tocotrienols. The abundance of α-tocopherol in the human body and the comparable efficiency of all vitamin E molecules as antioxidants, led biologists to neglect the non-tocopherol vitamin E molecules as topics for basic and clinical research. Recent developments warrant a serious reconsideration of this conventional wisdom. Tocotrienols possess powerful neuroprotective, anti-cancer and cholesterol lowering properties that are often not exhibited by tocopherols. Current developments in vitamin E research clearly indicate that members of the vitamin E family are not redundant with respect to their biological functions. α-Tocotrienol, γ-tocopherol, and δ-tocotrienol have emerged as vitamin E molecules with functions in health and disease that are clearly distinct from that of α-tocopherol. At nanomolar concentration, α-tocotrienol, not α-tocopherol, prevents neurodegeneration. On a concentration basis, this finding represents the most potent of all biological functions exhibited by any natural vitamin E molecule. An expanding body of evidence support that members of the vitamin E family are functionally unique. In recognition of this fact, title claims in manuscripts should be limited to the specific form of vitamin E studied. For example, evidence for toxicity of a specific form of tocopherol in excess may not be used to conclude that high-dosage “vitamin E” supplementation may increase all-cause mortality. Such conclusion incorrectly implies that tocotrienols are toxic as well under conditions where tocotrienols were not even considered. The current state of knowledge warrants strategic investment into the lesser known forms of vitamin E. This will enable prudent selection of the appropriate vitamin E molecule for studies addressing a specific need.
In 1905, Englishman William Fletcher determined that if special factors (vitamins) were removed from food disease ensued. Fletcher was researching the causes of the disease Beriberi when he discovered that eating unpolished rice prevented Beriberi and eating polished rice did not. William Fletcher believed that there were special nutrients contained in the husk of the rice. Next year, English biochemist Sir Frederick Gowland Hopkins also discovered that certain food factors were important to health. In 1912, Polish scientist Cashmir Funk named the special nutritional parts of food as a "vitamine" after "vita" meaning life and "amine" from compounds found in the thiamine he isolated from rice husks. Vitamine was later shortened to vitamin when it was discovered that not all of the vitamins contain nitrogen, and, therefore, not all are amines. Together, Hopkins and Funk formulated the vitamin hypothesis of deficiency disease - that a lack of vitamins could make people sick. Vitamin E was discovered in 1922 in green leafy vegetables by University of California researchers, Herbert Evans and Katherine Bishop. In 1924, Sure named it vitamin E. Because E supported fertility, it was scientifically named tocopherol. This comes from the Greek word tokos meaning childbirth, and phero meaning to bring forth, and the ol ending was added to indicate the alcohol properties of this molecule. In 1936 it was discovered that vitamin E was abundant in wheat germ oil. Two years later, it was chemically synthesized for the first time. The U.S. National Research Council sponsored studies on deficiencies of vitamin E, and based on the results E was designated an essential vitamin. Vitamin E emerged as an essential, fat-soluble nutrient that functions as an antioxidant in the human body. It is essential, because the body cannot manufacture its own vitamin E and foods and supplements must provide it. Since the elucidation of the chemical structure of vitamin E in 1938 by Fenholz and the synthesis of dl-α-tocopherol by Karrer in the same year, specific focus was directed on the chemical class of natural compounds that qualify to be vitamin E. At present, vitamin E represents a generic term for all tocopherols and their derivatives having the biological activity of RRR-α-tocopherol, the naturally occurring stereoisomer compounds with vitamin E activity (Traber and Packer, 1995; Traber and Sies, 1996). In nature, eight substances have been found to have vitamin E activity: α-, β-, γ- and δ-tocopherol; and α-, β-, γ- and δ-tocotrienol (Figure 1). Yet, of the 24000+ papers on vitamin E listed in PubMed, only just over 200 relate to tocotrienols (Table 1). The current handicap in knowledge of how tocotrienols may be implicated in human health and disease and the significance of filling that void in vitamin E research is discussed in this minireview.
Vitamin E are essential components of the human diet and are synthesized exclusively by photosynthetic organisms. Tocopherols consist of a chromanol ring and a 15-carbon tail derived from homogentisate (HGA) and phytyl diphosphate, respectively (Fig. 1). Condensation of HGA and phytyl diphosphate, the committed step in tocopherol biosynthesis, is catalyzed by HGA phytyltransferase (HPT). Tocotrienols differ structurally from tocopherols by the presence of three trans double bonds in the hydrocarbon tail (Fig. 1). Tocotrienols are the primary form of vitamin E in the seed endosperm of most monocots, including agronomically important cereal grains such as wheat, rice, and barley. Tocotrienols are also found in the seed endosperm of a limited number of dicots, including Apiaceae species and certain Solanaeceae species, such as tobacco. These molecules are found only rarely in vegetative tissues of plants. Crude palm oil extracted from the fruits of Elaeis guineensis particularly contains a high amount of tocotrienols (up to 800 mg/kg), mainly consisting of γ-tocotrienol and α-tocotrienol. Tocopherols, by contrast, occur ubiquitously in plant tissues and are the exclusive form of vitamin E in leaves of plants and seeds of most dicots. Transgenic expression of the barley HGGT (homogentisic acid transferase, which catalyzes the committed step of tocotrienol biosynthesis) in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux (Cahoon et al., 2003). Recently, another strategy involving genetic engineering of metabolic pathways in plants has proved to be efficient in bolstering tocotrienol biosynthesis (Rippert et al., 2004). In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. The flux of these two compounds has been upregulated by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast prephenate dehydrogenase gene in tobacco plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. Thus, the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants (Rippert et al., 2004).
Often, the term vitamin E is synonymously used with α-tocopherol. While the expression is correct it is incomplete and may be often misleading. d-α-Tocopherol (RRR-α-tocopherol) has the highest bioavailability and is the standard against which all the others must be compared. However, it is only one out of eight natural forms of vitamin E. Tocotrienols, formerly known as ζ, or η-tocopherols (Fig. 1), are similar to tocopherols except that they have an isoprenoid tail with three unsaturation points instead of a saturated phytyl tail (Fig. 1). Interestingly, tocotrienols possess powerful neuroprotective, antioxidant, anti-cancer and cholesterol lowering properties that often differ from the properties of tocopherols (Table 1). Micromolar amounts of tocotrienol suppress the activity of HMG-CoA reductase, the hepatic enzyme responsible for cholesterol synthesis (Pearce et al., 1994; Pearce et al., 1992). Tocotrienols are thought to have more potent antioxidant properties than α-tocopherol (Serbinova et al., 1991; Serbinova and Packer, 1994). The unsaturated side chain of tocotrienol allows for more efficient penetration into tissues that have saturated fatty layers such as the brain and liver (Suzuki et al., 1993). Experimental research examining the antioxidant, free radical scavenging effects of tocopherol and tocotrienols revealed that tocotrienols appear superior due to their better distribution in the fatty layers of the cell membrane (Suzuki et al., 1993). One major justification often used to side-line tocotrienol research is the relative inferiority of the bioavailability of orally taken tocotrienols compared to that of α-tocopherol. The hepatic α-tocopherol transfer protein (α-TTP), together with the tocopherol-associated proteins (TAP) is responsible for the endogenous accumulation of natural α-tocopherol. Although these systems have a much lower affinity to transport tocotrienols, it has been evident that orally supplemented tocotrienol results in plasma tocotrienol concentration in the range of 1 μM (O'Byrne et al., 2000). Of note, such circulating levels of α-tocotrienol are almost an order of magnitude higher than that required to protect neurons against a range of neurotoxic insults (Khanna et al., 2003; Sen et al., 2000). Despite such promising potential, tocotrienol research accounts for less than 1% of all vitamin E research published in PubMed. The unique vitamin action of α-tocopherol, combined with its prevalence in the human body and the similar efficiency of tocopherols as chain-breaking antioxidants, led biologists to almost completely discount the "minor" vitamin E molecules as topics for basic and clinical research. Recent discoveries have forced a serious reconsideration of this conventional wisdom (Hensley et al., 2004).
In 1950, Kamimura’s treatment of frostbite using α-tocopherol represents one of the earliest therapeutic applications of the vitamin (Kamimura, 1977). Early works of Tappel identified that α-tocopherol effectively inhibits biological oxidation processes (Tappel, 1953, 1954, 1955; Zalkin and Tappel, 1960; Zalkin et al., 1960). It was soon realized that tocopherol deficiency in humans led to elevated levels of oxidative lipid damage and erythrocyte hemolysis (Horwitt et al., 1956). These observations set the stage for the emergence of tocopherol as a biological antioxidant (Green and Bunyan, 1969), a concept that drew widespread attention in the decades to follow. Two decades after the “biological antioxidant theory” (Green and Bunyan, 1969) was reported, Burton and Ingold presented the first comprehensive review article discussing that α-tocopherol has near optimal activity as a chain-breaking antioxidant and that both the phenolic head and phytyl tails contributed to the biological properties of the vitamin E molecule (Burton and Ingold, 1989). α-Tocopherol gained recognition as the most important lipophilic radical-chain-breaking antioxidant in tissues in vivo. Deficiency of α-tocopherol in membranes made them highly permeable and therefore vulnerable to degradation. Tocopherols seemed also to influence other important biophysical membrane characteristics, such as fluidity, in a manner similar to that of cholesterol. Studies of the antioxidant properties led to the recognition that during the reaction of α-tocopherol with an appropriate oxidizing species, α-tocopherol may be oxidized to α-tocopheryl quinine (Seward et al., 1969). In latter studies where peroxidizing lipids were used to induce the formation of antioxidant radicals, electron spin resonance spectroscopy revealed that free radical interactions of dl-α-tocopherol generate dl-α-tocopheroxyl radicals. It was thus realized that α-tocopherol is only available as an antioxidant for a short period of time (Lambelet and Loliger, 1984). Importantly, it was noted that the reaction kinetics and stability of the four tocopherols were not identical. The fast reacting dl-α-tocopherol reacted more rapidly and trapped free radicals more thoroughly and was therefore only available as an antioxidant for a short period of time as compared with the slowly reacting dl-δ-tocopherol. dl-β- and dl-γ-Tocopherols behaved in an intermediate way (Lambelet and Loliger, 1984). That ascorbate can transfer hydrogen to α-tocopheroxyl radicals and thus regenerate α-tocopherol (Bascetta et al., 1983) encouraged the concept of antioxidant recycling. Mass analysis studies demonstrated that tocopherol can be regenerated in human cell homogenates implying that maintenance of membrane tocopherol status may be an essential function of ascorbate and GSH which operate in concert to ensure maximum membrane protection against oxidative damage (Chan et al., 1991). While the concept of antioxidant recycling was extended to build the “antioxidant network” hypothesis (Packer and Suzuki, 1993), skepticism regarding whether such interactions take place in vivo stirred the field (Strain and Mulholland, 1992). In the late eighties, the discovery that oxidative modification of low-density lipoprotein is a key trigger for atherosclerosis represented a major breakthrough in biomedical research. The early nineties was thus a time when numerous laboratories studied mechanisms underlying the oxidation of LDL and the inhibition of such oxidation. Because α-tocopherol was identified as the major antioxidant present in human lipoproteins, it received much attention as a suppressor of LDL lipid oxidation and as an epidemiological marker for ischemic heart disease. While most laboratories were excited about α-tocopherol preventing LDL oxidation, Stocker et al published conditions under which α-tocopherol may actually act as a pro-oxidant via the α-tocopheroxyl radical (Bowry et al., 1995). This direct link established between vitamin E chemistry and health outcomes drew significant attention underscoring the potential adverse effects of redox-active antioxidant nutrients. Although the relevance of the proposed antioxidant network remained to be proven in in vivo systems, enthusiasm for therapeutic regimens including multiple antioxidant members of the network soared (Albanes et al., 1996; DeCosse et al., 1989; Fuchs and Kern, 1998; Hartman et al., 1998; Liede et al., 1998; McKeown-Eyssen et al., 1988; Mireles-Rocha et al., 2002; Porkkala-Sarataho et al., 2000; Rapola et al., 1998; Rapola et al., 1997; Salonen et al., 2000; Teikari et al., 1998; Teikari et al., 1997; Woodson et al., 1999) resulting in quite a few clinical trials at a time when basic scientists were still trying to grasp the fundamentals.
All eight tocols in the vitamin E family share close structural similarity (Fig. 1) and hence comparable antioxidant efficacy (Table 1). Yet, current studies of the biological functions of vitamin E continue to indicate that members in the vitamin E family possess unique biological functions often not shared by other family members. One of the earliest observations suggesting that α-tocopherol may have functions independent of its antioxidant property came from the study of platelet adhesion. α-Tocopherol strongly inhibits platelet adhesion. Doses of 400 IU/day provide greater than 75% inhibition of platelet adhesion to a variety of adhesive proteins when tested at low shear rate in a laminar flow chamber. The antiadhesive effect of α-tocopherol appeared to be related to a reduction in the number and size of pseudopodia upon platelet activation and led to the hypothesis that within the body vitamin E may exert functions beyond its antioxidant property (Steiner, 1993). That members of the tocopherol family may have functions independent of their antioxidant properties gained more prominence when vitamin E molecules with comparable antioxidant properties exhibited contrasting biological effects (Boscoboinik et al., 1991). At the posttranslational level, α-tocopherol inhibits protein kinase C, 5-lipoxygenase and phospholipase A2 and activates protein phosphatase 2A and diacylglycerol kinase. Some genes (e.g. scavenger receptors, α-TTP, α-tropomyosin, matrix metalloproteinase-19 and collagenase) are specifically modulated by α-tocopherol at the transcriptional level. α-Tocopherol also inhibits cell proliferation, platelet aggregation and monocyte adhesion. These effects have been characterized to be unrelated to the antioxidant activity of vitamin E, and possibly reflect specific interactions of α-tocopherol with enzymes, structural proteins, lipids and transcription factors (Zingg and Azzi, 2004). γ-Tocopherol represents the major form of vitamin E in the diet in the USA, but not in Europe. Desmethyl tocopherols, such as γ-tocopherol and specific tocopherol metabolites, most notably the carboxyethyl-hydroxychroman (CEHC) products, exhibit functions that are not shared by α-tocopherol. The activities of these other tocopherols do not map directly to their chemical antioxidant behavior but rather reflect anti-inflammatory, antineoplastic, and natriuretic functions possibly mediated through specific binding interactions (Hensley et al., 2004). Metabolites of γ-tocopherol (2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman), but not that of α-tocopherol, provides natriuretic activity. Moreover, a nascent body of epidemiological data suggests that γ-tocopherol is a better negative risk factor for certain types of cancer and myocardial infarction than is α-tocopherol (Wagner et al., 2004). Further evidence supporting the unique biological significance of vitamin E family members is provided by current results derived from α-tocotrienol research. As illustrated in Table 1, α-tocotrienol possesses numerous functions that are not shared by α-tocopherol. For example, nanomolar concentrations of α-tocotrienol uniquely prevents inducible neurodegeneration by regulating specific mediators of cell death (Khanna et al., 2003; Sen et al., 2000). In addition, tocopherols do not seem to share the cholesterol-lowering properties of tocotrienol (Qureshi et al., 1986; Qureshi et al., 2002). Tocotrienol, not tocopherol, administration reduces oxidative protein damage and extends the mean life span of C. elegans (Adachi and Ishii, 2000). Furthermore, tocotrienol but not tocopherol, suppresses growth of human breast cancer cells (Nesaretnam et al., 1995). Such expanding body of evidence indicating that members of the vitamin E family are functionally unique calls for a revisit of the current practices in vitamin E research. Research claims should be limited to the specific form of vitamin E studied. For example, evidence for toxicity of a specific form of tocopherol in excess may not be used to conclude that high-dosage vitamin E supplementation may increase all-cause mortality (Miller et al., 2005). Along these lines, it may not be prudent to express frustrations about the net yield of vitamin E research as a whole (Greenberg, 2005) when all that has been tested for efficacy on a limited basis in clinical trials is α-tocopherol. Vitamin E represents one of the most fascinating natural resources that have the potential to influence a broad range of mechanisms underlying human health and disease. Yet, clinical outcomes studies have failed to meet expectations (Friedrich, 2004; Greenberg, 2005). The current state of knowledge warrants strategic investment into the lesser known forms of vitamin E with emphasis on uncovering the specific conditions that govern the function of vitamin E molecules in vivo. Outcomes studies designed in light of such information would yield lucrative returns.
Tocotrienol research in the laboratory is supported by NIH RO1NS42617 to CKS.