*3.1.2. Vitamin E intake*

Several key enzymes determine the rate of vitamin E catabolism (the reader referred to Section 2.4 "Hepatic metabolism of vitamin E") and, as outlined in the previous section, there is evidence that vitamin E in general might regulate its own metabolism. However, there are differences in the ability to regulate the metabolism depending on structural properties of the vitamin E isomers (i.e., methylation of the chroman ring, saturation of the side-chain, and stereochemistry). In principle, high intake of vitamin E, independent from the isomer, leads to enhanced formation of the respective metabolites [49]. However, the catabolic rates of the different forms of vitamin E clearly differ: the γ-isoforms are more susceptible to metabolization than the α-isoforms. Subjects supplemented with γ-T3 and α-T3 (125 mg or 500 mg) showed four to six times higher urinary excretion of the catabolic end product γ-CEHC and an induction of α-CEHC only after high dose (500 mg), but not after low dose supplementation (125 mg) [86]. In line with this, equimolar supplementation with 50 mg of α- and γ-TOH leads to a twofold increase of plasma γ-CEHC, but no alterations in α-CEHC [87]. These data indicate that there might be a threshold for the intake of α-TOH and α-T3 (or plasma levels, respectively) that needs to be exceeded to accelerate catabolism of α-TOH and α-T3 to form α-CEHC, as suggested by Schuelke et al. [88]. Interestingly, already in 1985, Handelman et al. reported that high α-TOH levels in human plasma are related to low γ-TOH levels [89]. After supplementation of α-TOH, the plasma α-TOH levels were, as expected, twofold to fourfold higher, but the γ-TOH level decreased to between one-third and one-half of the initial level [89]. Hence, α-TOH intake seems to boost γ-TOH catabolism. Supporting data were generated in a rat model, where the combined supplementation of α- and γ-TOH leads to higher excretion of γ-CEHC than the supplementation of γ-TOH alone [90], as well as the reported stimulation of γ-TOH catabolism by α-TOH in HepG2 liver cells [91]. Although the underlying mechanisms are not fully unraveled, there is evidence that α-TOH induces the activity of enzymes involved in the metabolism of vitamin E, leading to the degradation of non-α-forms, while α-TOH remains protected (please refer to Section 3.1.1 "Intracellular handling of vitamin E").

CYP3A4 [46]. In addition to the *in vitro* data, Uchida and coworkers investigated the inhibitory effects of sesamin on vitamin E metabolism in rats. Vitamin E-deficient rats (vitamin E free diet for 4 weeks) were treated with 50 mg/kg *RRR*-α-TOH alone or in combination with 200 g/kg sesame seeds [95]. Next, the concentration of α-TOH in different tissues as well as the urinary excretion of α-CEHC was measured. The urinary excretion of α-CEHC in the sesamin group was significantly lower compared to the α-TOH control group. Further, the combination of α-TOH and sesamin provoked a significant increase of hepatic α-TOH concentrations compared to α-TOH treated animals [95]. These observations have been confirmed in other animal studies [93, 94]. Beside the investigations in animal models, there are also a few results originating from studies in humans. In 2004, Frank and colleagues used muffins enriched with sesame oil (94 mg sesamin/muffin) or corn oil (control) to investigate the effect of a single dose sesamin application on urinary excretion of γ-CEHC as well as blood levels of γ-TOH in 10 healthy volunteers [97]. Both, control and intervention group, received the muffins together with a capsule containing deuterium-labeled γ-TOH (50 mg) in a crossover design. Blood and urine samples were collected over 72 hours after the application of the muffins and capsules. While the urinary excretion of γ-CEHC was significantly lowered, the sesamin treatment did not affect γ-TOH concentrations in blood compared to the corn oil control group [97]. Unfortunately, the study does not provide data on the elevation of the hepatic γ-TOH concentration in response to the reduced urinary excretion of γ-CEHC. Taken together, *in vitro* and *in vivo* studies provide evidence that the dietary intake of sesamin leads to an increase of the hepatic concentration of TOH via the inhibition of vitamin E metabolism, but further experiments are needed to charac-

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terize the interaction of sesamin and vitamin E metabolism in more detail.

The pharmacological modification of the enzymatic activity of CYP3A4 represents an effective way to influence vitamin E homeostasis in the human body. Mechanistically, the direct or indirect interference of vitamin E metabolism is usually just a side effect of the pharmacological inhibition or induction of CYP3A4 by various chemical compounds. Thus, it is not surprising that the first evidence for the involvement of CYP3A4 in vitamin E metabolism was provided in an experimental subset using ketoconazole as a specific inhibitor for CYP3A4 [46, 98]. In HepG2 liver cells, different concentrations of ketoconazole (1 mmol/l or 0.25 mmol/l) inhibited the metabolic conversion of γ- and δ-TOH (25 μmol/l cell culture media) to γ- or δ-CEHC by almost 90% [46]. This finding has been confirmed by the reproduction of the same experiment with sesamin, the natural inhibitor of CYP3A4, revealing comparable results [46]. The inhibitory effect of ketoconazole on vitamin E metabolism has further been observed in an *in vivo* model. Here, rats were supplemented with ketoconazole (50 mg/kg body weight) together with α-TOH (10 mg/kg body weight), γ-TOH (10 mg/kg body weight) or mixture of different T3s (29.5 mg/kg body weight). Ketoconazole significantly reduced the catabolism of all applied vitamin E forms resulting in impaired urinary excretion of the respective CEHCs [99]. Beside its inhibition, the pharmacological induction of CYP3A4 represents another way to modulate vitamin E metabolism. Birringer and coworkers demonstrated that 50 μmol/L rifampicin, an inducer of CYP3A4 activity [100], induced the degradation of all-*rac*-α-TOH

*3.2.2. Pharmacological activation or inhibition of CYP3A4*

## **3.2. Effects of other compounds**

### *3.2.1. Intake of sesamin*

Sesamin is a lignan, a group of natural compounds derived from vegetable sources, like sesame seeds [92]. Sesamin is known as a natural inhibitor of the metabolism of TOH [93–97]. The cell regulatory actions of sesamin have been initially investigated in *in vitro* models, where Parker et al. showed that sesamin acts as a selective inhibitor of CYP3A4, an initial enzyme of TOH metabolism [46]. In this study, the authors compared the inhibitory potential of sesamin on TOH metabolism in human HepG2 cells to the well-characterized CYP3A4 inhibitor ketoconazole. HepG2 cells were treated with one of the mentioned compounds in combination with either 25 μM α-TOH or 25 μM γ-TOH. Afterwards, the concentration of the corresponding CEHC was determined as a marker for TOH metabolism in cell culture media. It became apparent that ketoconazole (1 μM) and sesamin (1 μM) inhibited the formation of α- and γ-CEHC. This result provides evidence that sesamin is able to modulate TOH metabolism via the inhibition of CYP3A4 [46]. In addition to the *in vitro* data, Uchida and coworkers investigated the inhibitory effects of sesamin on vitamin E metabolism in rats. Vitamin E-deficient rats (vitamin E free diet for 4 weeks) were treated with 50 mg/kg *RRR*-α-TOH alone or in combination with 200 g/kg sesame seeds [95]. Next, the concentration of α-TOH in different tissues as well as the urinary excretion of α-CEHC was measured. The urinary excretion of α-CEHC in the sesamin group was significantly lower compared to the α-TOH control group. Further, the combination of α-TOH and sesamin provoked a significant increase of hepatic α-TOH concentrations compared to α-TOH treated animals [95]. These observations have been confirmed in other animal studies [93, 94]. Beside the investigations in animal models, there are also a few results originating from studies in humans. In 2004, Frank and colleagues used muffins enriched with sesame oil (94 mg sesamin/muffin) or corn oil (control) to investigate the effect of a single dose sesamin application on urinary excretion of γ-CEHC as well as blood levels of γ-TOH in 10 healthy volunteers [97]. Both, control and intervention group, received the muffins together with a capsule containing deuterium-labeled γ-TOH (50 mg) in a crossover design. Blood and urine samples were collected over 72 hours after the application of the muffins and capsules. While the urinary excretion of γ-CEHC was significantly lowered, the sesamin treatment did not affect γ-TOH concentrations in blood compared to the corn oil control group [97]. Unfortunately, the study does not provide data on the elevation of the hepatic γ-TOH concentration in response to the reduced urinary excretion of γ-CEHC. Taken together, *in vitro* and *in vivo* studies provide evidence that the dietary intake of sesamin leads to an increase of the hepatic concentration of TOH via the inhibition of vitamin E metabolism, but further experiments are needed to characterize the interaction of sesamin and vitamin E metabolism in more detail.

### *3.2.2. Pharmacological activation or inhibition of CYP3A4*

*3.1.2. Vitamin E intake*

8 Vitamin E in Health and Disease

**3.2. Effects of other compounds**

*3.2.1. Intake of sesamin*

Several key enzymes determine the rate of vitamin E catabolism (the reader referred to Section 2.4 "Hepatic metabolism of vitamin E") and, as outlined in the previous section, there is evidence that vitamin E in general might regulate its own metabolism. However, there are differences in the ability to regulate the metabolism depending on structural properties of the vitamin E isomers (i.e., methylation of the chroman ring, saturation of the side-chain, and stereochemistry). In principle, high intake of vitamin E, independent from the isomer, leads to enhanced formation of the respective metabolites [49]. However, the catabolic rates of the different forms of vitamin E clearly differ: the γ-isoforms are more susceptible to metabolization than the α-isoforms. Subjects supplemented with γ-T3 and α-T3 (125 mg or 500 mg) showed four to six times higher urinary excretion of the catabolic end product γ-CEHC and an induction of α-CEHC only after high dose (500 mg), but not after low dose supplementation (125 mg) [86]. In line with this, equimolar supplementation with 50 mg of α- and γ-TOH leads to a twofold increase of plasma γ-CEHC, but no alterations in α-CEHC [87]. These data indicate that there might be a threshold for the intake of α-TOH and α-T3 (or plasma levels, respectively) that needs to be exceeded to accelerate catabolism of α-TOH and α-T3 to form α-CEHC, as suggested by Schuelke et al. [88]. Interestingly, already in 1985, Handelman et al. reported that high α-TOH levels in human plasma are related to low γ-TOH levels [89]. After supplementation of α-TOH, the plasma α-TOH levels were, as expected, twofold to fourfold higher, but the γ-TOH level decreased to between one-third and one-half of the initial level [89]. Hence, α-TOH intake seems to boost γ-TOH catabolism. Supporting data were generated in a rat model, where the combined supplementation of α- and γ-TOH leads to higher excretion of γ-CEHC than the supplementation of γ-TOH alone [90], as well as the reported stimulation of γ-TOH catabolism by α-TOH in HepG2 liver cells [91]. Although the underlying mechanisms are not fully unraveled, there is evidence that α-TOH induces the activity of enzymes involved in the metabolism of vitamin E, leading to the degradation of non-α-forms, while α-TOH

remains protected (please refer to Section 3.1.1 "Intracellular handling of vitamin E").

Sesamin is a lignan, a group of natural compounds derived from vegetable sources, like sesame seeds [92]. Sesamin is known as a natural inhibitor of the metabolism of TOH [93–97]. The cell regulatory actions of sesamin have been initially investigated in *in vitro* models, where Parker et al. showed that sesamin acts as a selective inhibitor of CYP3A4, an initial enzyme of TOH metabolism [46]. In this study, the authors compared the inhibitory potential of sesamin on TOH metabolism in human HepG2 cells to the well-characterized CYP3A4 inhibitor ketoconazole. HepG2 cells were treated with one of the mentioned compounds in combination with either 25 μM α-TOH or 25 μM γ-TOH. Afterwards, the concentration of the corresponding CEHC was determined as a marker for TOH metabolism in cell culture media. It became apparent that ketoconazole (1 μM) and sesamin (1 μM) inhibited the formation of α- and γ-CEHC. This result provides evidence that sesamin is able to modulate TOH metabolism via the inhibition of The pharmacological modification of the enzymatic activity of CYP3A4 represents an effective way to influence vitamin E homeostasis in the human body. Mechanistically, the direct or indirect interference of vitamin E metabolism is usually just a side effect of the pharmacological inhibition or induction of CYP3A4 by various chemical compounds. Thus, it is not surprising that the first evidence for the involvement of CYP3A4 in vitamin E metabolism was provided in an experimental subset using ketoconazole as a specific inhibitor for CYP3A4 [46, 98]. In HepG2 liver cells, different concentrations of ketoconazole (1 mmol/l or 0.25 mmol/l) inhibited the metabolic conversion of γ- and δ-TOH (25 μmol/l cell culture media) to γ- or δ-CEHC by almost 90% [46]. This finding has been confirmed by the reproduction of the same experiment with sesamin, the natural inhibitor of CYP3A4, revealing comparable results [46]. The inhibitory effect of ketoconazole on vitamin E metabolism has further been observed in an *in vivo* model. Here, rats were supplemented with ketoconazole (50 mg/kg body weight) together with α-TOH (10 mg/kg body weight), γ-TOH (10 mg/kg body weight) or mixture of different T3s (29.5 mg/kg body weight). Ketoconazole significantly reduced the catabolism of all applied vitamin E forms resulting in impaired urinary excretion of the respective CEHCs [99]. Beside its inhibition, the pharmacological induction of CYP3A4 represents another way to modulate vitamin E metabolism. Birringer and coworkers demonstrated that 50 μmol/L rifampicin, an inducer of CYP3A4 activity [100], induced the degradation of all-*rac*-α-TOH in HepG2 cells fivefold [47]. In this study, the cell culture medium has been preconditioned with 100 μmol/L α-TOH for 10 days, as the standard medium was deficient for α-TOH [47]. Further, an indirect approach for the modulation of vitamin E metabolism via the modification of CYP3A4 expression could be realized by triggering PXR, a nuclear receptor that regulates the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics [101, 102]. Landes and coworkers showed that γ-T3 as well as rifampicin acts as PXR agonists, thus upregulating CYP3A4 mRNA expression in HepG2 liver cells [81]. Given the fact that enhanced mRNA expression of CYP3A4 results in enhanced enzymatic activity, the stimulation of PXR by various pharmacological agonists or antagonists could also modulate the hepatic metabolism of vitamin E. In summary, the direct or indirect regulation of CYP3A4 by various pharmacological means represents an effective way to modify the hepatic vitamin E metabolism.

is thought to counteract increased oxidative stress during aging. In the liver and heart, however, data are conflicting: while some found increased concentrations [37, 119, 120], Takahashi et al. found decreased values [117]. Two studies also analyzed the age-dependent regulation of genes, known to be involved in vitamin E handling, which are α-TTP, ABCA1, and Cyp4f14 (murine orthologue of CYP4F2) [117] as well as NPC1, NPC2, and LPL [37]. Takahashi et al. found increasing (mice with the age of 3–12 month) and then decreasing (12—24 months) α-TTP protein levels in the liver, while mRNA expression was stable over age [117]. Overall, Cyp4f14 mRNA expression decreased during aging (60% decrease in mRNA expression at the age of 24 months compared to the age of 3 weeks), while ABCA1 mRNA expression slightly increased (20% in the same age range as measured for Cyp4f14) [117]. The authors concluded that the age-related changes of hepatic α-TOH levels cannot be explained by the metabolism of α-TOH via Cyp4f14. König et al. analyzed protein expression in kidney tissue or its lysosomal membranes and found a significant decrease of NPC1 and NPC2, but a prominent increase in LPL (361% compared with the tissue from younger mice) [37]. The increased expression of LPL may explain the accumulation of α-TOH in aged mice. Furthermore, NPC1 and NPC2 may be responsible for the transport of α-TOH from the endosomes to the cytosol [69] and their reduced expression may explain the accumulation of α-TOH in lysosomal membranes [37]. In summary, there are age-dependent changes in α-TOH tissue and plasma concentrations and also in the expression of genes responsible for vitamin E handling; however, the

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The sex-dependent differences in vitamin E handling were described recently by Schmölz et al. [6] and will be summarized here briefly for humans only. While intake of vitamin E in total is higher in men than in women [122], the intake per kcal is higher for women than for men [123]. The absorption of α-TOH seems not to be influenced by sex, but is mainly regulated by downstream regulatory processes (likely by hepatic sorting or metabolism) [113]. The data on serum concentrations of vitamin E are inconsistent: while some researchers reported elevated α-TOH serum concentrations for women compared to men [124, 125], others found contradictory results [123]. Sex-dependent regulation of vitamin E metabolism is specific for the different forms of vitamin E. Women degrade γ-TOH to a higher degree than men, while the metabolism of α-TOH seems to be independent [87]. Two mechanisms may be relevant for sex-dependent regulation of vitamin E metabolism: the hormonal status of individuals and the activation of the CYP enzymes involved in vitamin E metabolism [6]. Further studies could illuminate gender-specific differences in more detail. In the light of the discovery of

The influence of genetics on vitamin E handling was summarized in detail in a recent review (for more details, please see [6]). Therefore, only a short overview will be provided here. Interindividual differences in the handling of vitamin E can be caused by individual genetic constitutions. Polymorphisms in genes, which are responsible for vitamin E handling such as

underlying regulatory processes are not unraveled completely yet.

vitamin E as a factor that limits female fertility, this is of special interest.

*3.3.2. Gender*

*3.3.3. Genetics*
