**2.5. Release of vitamin E**

that vitamin E also colocalizes with poly-unsaturated fatty acids (PUFAs) in nonraft domains in order to provide protection from lipid peroxidation [41]. Newly added α-TOH in cell culture enriches in the same organelles as the endogenous α-TOH pool [42]. Hereby, the subcel-

Our knowledge about the storage of vitamin E in lipid droplets is also limited. It was recently reported that newly endocytosed vitamin E was also found in lipid droplets, thus indicating

The hepatic metabolism of vitamin E has not been fully characterized. However, the principle steps of vitamin E degradation, that is, the shortening of the side chain without the alteration of the chroman ring, are generally accepted. Hence, the metabolites are classified as α-, β-, γ-,

In principle, TOHs and T3s are degraded like long branched chain fatty acids (TOH) or long unsaturated branched chain fatty acids (T3) via β-oxidation in peroxisomes. However, as TOHs and T3s do not bear a terminal carboxy function in their side chain, they are not susceptible to β-oxidation. Hence, the initial and rate-limiting step in vitamin E degradation is the introduction of a carboxy function to the ω-terminus of the side chain. This first step is carried out in the endoplasmic reticulum (ER) of liver cells [44]. Here, two representatives of the cytochrome P450 (CYP) protein family, namely, CYP4F2 [45] and CYP3A4 [46, 47], have been identified to catalyze the initial ω-hydroxylation step. The resulting 13′-hydroxychromanol (13′-OH) is then further metabolized via ω-oxidation, a step that most likely involves alcohol dehydrogenase and aldehyde dehydrogenase [44], leading to 13′-carboxychromanol (13′-COOH). The carboxylated side chain resembles a long branched chain fatty acid that is further degradable via β-oxidation. However, a transport mechanism for the carboxychromanol from the ER to the peroxisomes has not been identified so far. Nevertheless, two cycles of peroxisomal β-oxidation after the activation of α-13′-COOH to the respective CoA ester have been suggested [44], as the peroxisomal β-oxidation system has a higher affinity toward long branched chain fatty acids than the mitochondrial counterpart [48]. The proposed 11′- and 9′-COOH metabolites have indeed been identified in human and mouse samples [49] as well as in a hepatic cell line [45, 50]. Subsequently, three more cycles of β-oxidation are needed to form the final product of vitamin E degradation, namely, carboxyethyl hydroxychromanol (CEHC) or 3′-COOH. These steps, however, are assigned to mitochondrial β-oxidation, as CEHC has solely been found in the mitochondria of hepatic cells [44]. Again, the transport mechanisms of the long-chain metabolites (LCM) (13′- to 9′-COOHs) from peroxisomes to the mitochondria are not known. The respective products for each cycle of β-oxidation (7′-COOH, 5′-COOH, and 3′-COOH) have been identified in different human and murine tissues [49, 51–54] as well as the hepatic cell line HepG2 [45, 47, 51]. Taken together, the hepatic metabolism of vitamin E is characterized by a series of β-oxidation steps after an initial introduction of a carboxy moiety at the ω-terminus of the phytyl-like side chain. The metabolism likely takes place in different cell compartments depending on the enzymatic systems needed for the different degradation steps. However, a concept of vitamin E degradation exclusively in mitochondria cannot be excluded [44]. T3 degradation is believed to follow the same route as TOH degradation but requiring further steps due to the unsaturated side

lular content of α-TOH was directly proportional to the lipid content [43].

endosome-lipid-droplet interactions [33].

and δ-metabolites according to their respective precursors.

**2.4. Hepatic metabolism of vitamin E**

4 Vitamin E in Health and Disease

Following the nature of the lipoprotein metabolism, hepatic release of vitamin E is mostly realized via VLDL. Thus, this section will focus on the packaging of vitamin E into VLDL particles, notwithstanding that the mechanism is not well understood. However, hepatic transfer of vitamin E to HDL has also been suggested [56]. Since it was shown that the expression of α-TTP is crucial for the maintenance of plasma α-TOH levels [57, 58] and that the liver is controlling plasma α-TOH levels [59], hepatic α-TTP is likely involved in the incorporation of vitamin E into lipoproteins. This concept is supported by the observation that nascent VLDL particles are preferentially enriched with *RRR*-α-TOH after oral administration of vitamin E ([60, 61]. In contrast, in the liver, no preferential retention of *RRR*-α-TOH was found, indicating that α-TTP is not involved in the delivery of vitamin E to the liver, but in the release from the liver [62]. Hence, efforts have been made to identify the intracellular location of VLDL enrichment with α-TOH mediated by α-TTP [30]. According to the assembly of VLDL, either the rough ER or the Golgi apparatus were assumed. However, the action of α-TTP in these compartments was not confirmed as the nascent VLDL particles contained equal amounts of SRR and RRR α-TOH forms [30]. Further, the inhibition of ER/Golgi action in cells overexpressing α-TTP did not prevent α-TOH secretion [63]. In conclusion, α-TTP is necessary for the hepatic release of vitamin E, but the enrichment of VLDL with *RRR*-α-TOH occurs after exocytosis.

Based on this, the hypothesis of α-TOH uptake by VLDL directly from the plasma membrane was developed. This idea was inspired by the proposed mechanism of the incorporation of free cholesterol into nascent VLDL [64], that is, the spontaneous transfer from membranes to lipoproteins [65]. The hypothesis involves also the α-TTP-mediated trafficking of vitamin E from late endosomes (where vitamin E occurs after cellular uptake and large parts of α-TTP are located [66]) to the plasma membrane. This process might involve ABCA1, which has been shown to transport α-TOH [67] and could thus present vitamin E to α-TTP at the outer leaflet of the endosomal membrane. After the transport to the plasma membrane, a yet unidentified flippase is required to transfer α-TOH to the appropriate site of the membrane for uptake by nascent VLDL [30]. This hypothesis is supported by findings of Chung et al. [33], which provided a model of α-TTP-facilitated trafficking of vitamin E from endosomes to the plasma membrane (the reader is referred to Section 2.2 "Intracellular trafficking of vitamin E"). Taken together, the release of α-TOH from hepatocytes depends on vesicular transport [21, 31, 63, 68, 69], but is independent from ER or Golgi [63]. Hence, lipoproteins are not loaded with TOH during their intracellular assembly, but rather after exocytosis, a mechanism is required for the presentation of α-TOH at the plasma membrane. Evidence has been provided that the trafficking of α-TOH to the plasma membrane is realized via α-TTP which is located at recycling endosomes in hepatocytes [33]. However, the mechanism of the loading of lipoproteins with α-TOH from the plasma membrane has not been elucidated yet, although the involvement of ABC transporters has been suggested [56, 67, 70]. However, ABC transporters are fueling HDL particles, which is in contrast to the assumption that the hepatic release of α-TOH is mediated via VLDL. In turn, two explanations have evolved: first, α-TOH translocates spontaneously from the membrane to VLDL like free cholesterol [65], and second, α-TOH is transported to HDL via ABCA1 and is then spontaneously transferred to VLDL [71]. However, both hypotheses need evaluation. A recent report on the self-assembly of α-TTP to form nanoparticles and transport vitamin E to tissues protected by endothelial barriers like the brain [34] opens another possible way for the distribution of vitamin E throughout the body starting from the liver.

report elevated α-TTP levels due to a higher intake of vitamin E [72, 77], but some revealed no effect [74–76] or even lower levels [73]. Hence, further studies are needed to clarify the role of vitamin E in the regulation of α-TTP. In addition, an *in vitro* study suggested that vitamin E does not regulate α-TTP at the level of gene expression, but stabilizes α-TTP at the protein level upon binding and thus protects the protein from degradation, leading to higher α-TTP protein levels [78]. Reports on the hepatic mRNA levels might thus be of minor importance for the interpretation of the contribution of vitamin E to α-TTP action; however, the findings

The Hepatic Fate of Vitamin E

7

http://dx.doi.org/10.5772/intechopen.79445

The rate-limiting enzymes of vitamin E metabolism are CYP4F2 and CYP3A4. The latter was reported to be under transcriptional control of pregnane-X-receptor (PXR) [79, 80]. Hence, vitamin E might regulate its metabolism by binding to PXR and subsequent alteration of the expression of the enzymes involved in the first catabolic step. Indeed, studies using cells transfected with reporter genes provided evidence for an activation of PXR by different vitamin E structures (i.e., TOHs, T3s, and metabolites) [81, 82]. Interestingly, α-, δ-, and γ-TOH as well as α- and γ-T3 activated PXR in HepG2 liver cells transfected with human PXR and chloramphenicol acetyl transferase linked to two PXR responsive elements [81], while α- and γ-TOH as well as their metabolites α- and γ-CEHC did not in transfected colon carcinoma cells [82]. However, the LCM α-13′-COOH activated PXR in the latter cellular system and so did γ-T3 [82]. This finding implicates that the LCM of TOH are the responsible mediators of reported TOH actions via PXR. Hence, the findings in hepatic HepG2 cells [81] might be due to a higher catabolic rate of TOH and in turn the more efficient formation of the LCM than in colon cells. However, these findings were made in artificial cellular reporter systems and might not resemble the actual (hepatic) situation *in vivo*. Further, the specificity of PXR might depend on the species, as γ-T3 (the vitamin E form that activated PXR in both of the aforementioned studies) fails to bind murine PXR [83]. However, results obtained *in vivo* support the regulation of Cyp3a11 (the murine orthologue of CYP3A4) by vitamin E via PXR. Mice supplemented with α-TOH show elevated hepatic expression of Cyp3a11, while their PXRdeficient counterparts as well as mice with humanized PXR showed no upregulation of Cyp3a11 in response to α-TOH [84]. The same finding was made for Cyp4f13, the murine orthologue of CYP4F2, in this model [84]. These findings suggest that both enzymes are under the control of PXR and murine, but not human PXR is susceptible to α-TOH (or its metabolites as outlined above). Further studies reporting upregulation of hepatic Cyp3a in rodent models with α-TOH supplementation support this finding [76, 83, 85]. Interestingly, in these studies, γ-TOH and γ-T3 had no effect on Cyp3a expression [83, 85], supporting the suggested specificity of murine PXR for α-TOH. In line with this, γ-TOH did not alter the expression of Cyp4f13 in mice [85]. However, subcutaneous application of α-TOH in rats did not induce Cyp4F2 levels [76], which is in contrast to the above mentioned induction of Cyp4f13 in mice via PXR [84]. The reported induction of CYP4F2 activity in HepG2 cells by α-TOH further complicates the interpretation of the data on the effect of vitamin E on CYP4F2 [45]. Taken together, there is evidence for the regulation of CYP4F2 and CYP3A4 via PXR by vitamin E in the human liver. However, several aspects need further clarification, for instance, species and vitamin E isoform specificity of PXR, the regulation of CYP4F2 by vitamin E or the relevance

on α-TTP protein expression are also inconsistent.

of the α-LCM as true mediators of α-TOH effects via PXR.
