In the current study we have used SQ injected α-tocopherol in rats to identify the subcellular location of α-tocopherol ω-hydroxylation and β-oxidation. Importantly, our data indicates that as liver α-tocopherol increases 1) there is a greater partitioning of α-tocopherol to the microsomes, as compared to peroxisomes or mitochondria and 2) microsome 13′-OH-α-tocopherol levels increase sharply, and to a much greater degree than 13′-OH-α-tocopherol levels in mitochondria or peroxisome fractions of the same liver. These data support earlier in vitro data indicating a key role for microsomes in α-tocopherol ω-hydroxylation in vivo.
Trolox, a structural analog of α-CEHC, was taken up by the liver and ~100% of the liver trolox was recovered in the liver homogenates. However, trolox concentraitons were below levels of detection in all subcellular fractions, indicating that trolox is not taken up by microsomes, peroxisomes or mitochondria. These data suggest that α-CEHC found in the subcellular fractions is not the result of uptake of α-CEHC produced elsewhere in the cell.
To our knowledge α-CEHC, the final product of α-tocopherol β-oxidation, has not previously been determined in hepatic subcellular fractions, nor have the enzymes involved in α-tocopherol β-oxidation been identified. Even so, the accepted paradigm in the literature is that α-tocopherol β-oxidation occurs exclusively in the peroxisomes [14
]. Hence our finding that α-CEHC is localized to the mitochondria, not the peroxisomes, is highly significant as it indicates that α-tocopherol β-oxidation does not occur exclusively in peroxisomes, but rather mitochondria are responsible for the final step(s) in α-tocopherol β-oxidation. The presence of α-CEHC exclusively in the mitochondrial fraction was true in animals supplemented and not supplemented with α-tocopherol, thus mitochondria play a role in α-tocopherol β-oxidation at both basal dietary and high hepatic levels of α-tocopherol.
In order for tocopherols to enter the β-oxidation pathway of either peroxisomes or mitochondria the side chain must undergo ω-hydroxylation to form a 13′-hydroxyl group at the end of the phytyl tail. This terminal hydroxyl group would need to be converted to an aldehyde and then a carboxylic acid. Enzymes capable of catalyzing these reactions are located in the endoplasmic reticulum and cytoplasm [23
]. Once converted to a carboxylic acid the tocopherol tail would structurally resemble a 2-methyl-branched fatty acid, i.e., pristanic acid (Figures and
). The next step would be activation to an acyl-CoA ester followed by β-oxidation.
Peroxisomal enzymes have high activity toward medium and long branched chain acyl-CoAs [16
], while mitochondrial β-oxidation enzymes have low activity [23
]. However, peroxisomal acyl-CoA-oxidases show very low activity towards short chain substrates [23
]. Thus, very long chain fatty acids and 2-methyl-branched fatty acids initially undergo one or more cycles of β-oxidation in peroxisomes and then are either hydrolyzed to free acids by peroxisomal thioesterases or transesterified to carnitine esters by enzymes present in the peroxisomes. The resulting products are then exported from the peroxisome, the free fatty acids are esterified with carnitine, and the carnitine esters are imported by the mitochondria where further rounds of β-oxidation occur (), as reviewed in [15
Based on our findings and the structural similarities between the 2-methyl-branched fatty acids and the phytyl tail of tocopherols, we propose that α-tocopherol β-oxidation, as well as β-oxidation of other tocopherols, utilizes the same enzymatic pathway as the 2-methyl-branched fatty acids. Accordingly, once converted to the carboxylic acid, tocopherols would undergo 2 cycles of peroxisomal β-oxidation utilizing enzymes known to β-oxidize 2-methyl-branched fatty acids, followed by 3 cycles of β-oxidation in the mitochondria, again utilizing the 2-methyl-branched fatty acid β-oxidation pathway (). Conversely, our data does not exclude a model in which the mitochondria are exclusively responsible for α-tocopherol β-oxidation.
Proposed pathway of α-tocopherol metabolism in which both peroxisomes and mitochondria play a role
Similar to α-tocopherol, the subcellular location of β-oxidation of other forms of vitamin E, including γ-tocopherol, has not been identified. However, sulfated metabolic intermediates of γ-tocopherol have been identified in the livers of rats supplemented with mega doses of γ-tocopherol [25
]. The presence of sulfated intermediates is not easily explained in a model of exclusive peroxisomal β-oxidation in which tocopherols would be expected to remain in the peroxisomes until fully metabolized to CEHC. On the other hand, in our proposed model of tocopherol metabolism, specific γ-tocopherol intermediates may accumulate in the cytoplasm as they move from the peroxisomes to the mitochondria. Cytoplamic sulfotransferase enzymes may then catalyze sulfation of the intermediates to increase their solubility and allow excretion, thus preventing accumulation of γ-tocopherol intermediates. Sulfated metabolic intermediates of α-tocopherol have not to date been identified in vivo, nor were we able to identify sulfated metabolic intermediates of α-tocopherol in the current study (Leonard, S. et al unpublished data). Hence, further studies are needed to fully elucidate the complete metabolic pathway of other forms of vitamin E, as well as α-tocopherol, under conditions of both basal and elevated intakes.
We previously demonstrated that daily SQ α-tocopherol injections (10 mg /100 g body wt) significantly increased hepatic 13′-OH-α-tocopherol and α-CEHC levels indicating that the liver was able to up-regulate α-tocopherol metabolism to prevent accumulation of “excess” α-tocopherol [4
]. To test the hypothesis that increased hepatic α-tocopherol metabolite levels may play a role in up-regulating pathways to prevent the accumulation of α-tocopherol and its metabolites we injected rats daily with trolox (10 mg /100 g body wt), an analog of α-CEHC with a side chain just one carbon shorter than α-CEHC (). However, elevated hepatic levels of trolox did not alter the hepatic levels α-tocopherol or α-tocopherol metabolites, nor did it alter the subcellular distribution of hepatic α-tocopherol or α-tocopherol metabolites. These data suggest that elevated hepatic α-CEHC does not alter α-tocopherol metabolism or that trolox is not a good analogue for studies of metabolism given its shorter tail.
In conclusion, SQ α-tocopherol injections increase hepatic microsome α-tocopherol levels to a greater degree than either mitochondria or peroxisome α-tocopherol levels suggesting a greater partitioning of α-tocopherol to the microsomes with increasing hepatic α-tocopherol. 13′-OH-α-Tocopherol was also selectively increased in the microsome fraction with increased hepatic α-tocopherol. Together these data (1) support the hypothesis that microsomes are responsible for the ω-hydroxylation of α-tocopherol and (2) suggest mechanisms are in place to selectively distribute α-tocopherol to the microsomes under conditions of elevated hepatic α-tocopherol, thus facilitating increased metabolism.
An important and unexpected finding of this study is that α-CEHC is not detectable in the hepatic peroxisome fraction but rather α-CEHC is localized to the mitochondrial fraction. Thus our data is the first to indicate a role for mitochondria in α-tocopherol β-oxidation. We propose that α-tocopherol β-oxidation utilizes the 2-methyl-branched chain β-oxidation pathway that includes enzymes located in both peroxisomes and mitochondria. Further elucidation of the specific metabolic enzymes and hepatic transporters involved in the regulation of α-tocopherol levels, as well as the ability of α-tocopherol to modulate its own metabolism and/or the metabolism and excretion of pharmaceutical drugs and environmental toxins, are needed in order that α-tocopherol supplements may be used with optimal health benefits, while avoiding possible adverse effects.