Gender differences in the inhibition of γ-tocopherol metabolism by a single dose of dietary sesame oil in healthy subjects

Abstract

Background:

γ-Tocopherol (γ-T) has unique properties that may be beneficial in sustaining optimal human health, but hepatic vitamin E metabolism enhances γ-T turnover.

Objective:

Our aim was to determine the extent to which dietary sesame lignans alter human α- and γ-T metabolism and elimination as carboxyethyl hydroxychromanols (CEHCs).

Design:

Healthy participants (n=5, each men and women) in a randomized, crossover study (4 w washout) consumed muffins prepared with either corn oil or unrefined sesame oil (sesamin, 94 mg; sesamolin, 42 mg), along with a capsule containing a 1:1 molar ratio of deuterium-labeled d₆-α- and d₂-γ-T acetates (~50 mg each). Plasma and urine were collected up to 72 h; unlabeled and labeled T and CEHC concentrations were determined by LC-MS.

Results:

Sesame oil muffin consumption in men, but not in women, decreased (P<0.05) areas under plasma d₂-γ-CEHC concentration-time curves (AUCs) and maximum concentrations. However, in both genders urinary d₂-γ-CEHCs were decreased for 24 h following sesame oil muffin consumption.

Conclusion:

In humans, γ-T metabolism can be inhibited by the simultaneous consumption of γ-T and sesame lignans. The observed differences between men and women with respect to vitamin E metabolism warrant further investigation.

INTRODUCTION

γ-Tocopherol (γ-T) has unique properties that may be beneficial in sustaining optimal human health and preventing disease (1). For example, γ-T as a result of the unsubstituted 5-position on the chromanol ring can scavenge reactive nitrogen species (RNS) (2). RNS damage in humans has been substantiated by the detection of higher 5-nitro-γ-T concentrations in smokers (3) and Alzheimer patients (4). γ-T reportedly inhibits thrombogenesis (5), decreases inflammation (6), as well as reduces cancer cell proliferation (7,8). Moreover, γ-T, but not α-T levels, were reduced in coronary heart disease patients (9-11), suggesting that there may be benefits in raising plasma γ-T concentrations (5,12).

Although the liver expresses the α-T transfer protein (α-T TP), which is responsible for salvaging α-T from the excretory pathway and returning to the liver, hepatic metabolism appears to be a key factor in the discrimination between tocopherols (13). In the fruit fly Drosophila melanogaster because the fly lacks α-TTP, the selective accumulation of α-T has been attributed to metabolism of non-α-Ts (14). Vitamin E forms are metabolized to side-chain truncated, water-soluble carboxyethyl hydroxychromans (CEHCs) (15-18) and are excreted in urine (19-21) and bile (22,23). Cytochrome P₄₅₀ (CYP) enzymes catalyze the initial ω-hydroxylation of the side-chain prior to its shortening by enzymes of the β-oxidation pathway (17,18). Importantly, these processes have a higher catalytic activity towards the non-α-vitamers (15,17).

Sesamin, an abundant lignan in sesame seeds and oils, when fed to rats substantially increased plasma γ-T, but not α-T concentrations (24,25). In HepG2 cells and primary rat hepatocytes, sesamin inhibited the degradation of non-α-Ts to their corresponding CEHC metabolites (18,26). Consistently, dietary sesame seeds or sesame lignans (sesamin and sesaminol) reduced the urinary excretion of γ-CEHC in rats (21). Increased blood γ-T concentrations in response to sesame lignan intake have also been reported in two controlled human studies (27,28). Thus, the lack of γ-T retention in blood is greatly influenced by hepatic metabolism.

Our previous studies using deuterium-labeled α- and γ-Ts demonstrated that not only does γ-T turnover faster than does α-T, but that γ-CEHC and γ-T disappear from the plasma at the same rates (29). Moreover, women had a faster γ-T disappearance rate than did men. These data suggested that vitamin E metabolism is critical in regulating plasma γ-T concentrations in humans. To evaluate the hypothesis that the simultaneous consumption of vitamin E with sesame oil containing sesame lignans could would decrease vitamin E metabolism, and thus increase plasma γ-T concentrations, a randomized, cross-over study was carried out in both men and women. The participants consumed deuterium labeled α- and γ-tocopheryl acetates along with a breakfast containing either sesame or corn oil muffins; after a 4 week washout, the subjects repeated the study with the opposite muffin.

METHODS

Materials

α-5,7-(C²H₃)₂ tocopheryl acetate (d₆-α-TAc) was a gift from Dr. James Clark of Cognis Nutrition and Health (LaGrange, IL, USA). γ-3,4-(²H) tocopheryl acetate (d₂-γ-TAc) was prepared from γ-T as described (30). The d₆-α- and d₂-γ-TAc were diluted in tocopherol-stripped corn oil at a 1:1 molar mixture, and gelatin capsules containing approximately 50 mg of each α- and γ-TAc, were prepared. The d₆-α- to d₂-γ-T molar ratio was determined by liquid chromatography/mass spectrometry (LC/MS) to be 0.98. The internal standard, α-5,7,8-(C²H₃)₃ tocopheryl acetate (d₉-α-TAc), was provided by Dr. Carolyn Good of The Bell Institute of Health and Nutrition (Minneapolis, MN, USA), and was synthesized by Isotec, Inc. (Miamisburg, OH, USA). Cold-pressed, unrefined sesame oil (a gift from Henry Lamotte GmbH, Bremen, Germany) contained sesamin, 607 mg/100 g oil and sesamolin, 269 mg/100 g oil. For use as standards 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman (α-CEHC) and 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (γ-CEHC) were gifts from W.J. Wechter of Loma Linda University (Loma Linda, CA, USA). Trolox, γ-T, ascorbic acid, butylated hydroxy toluene (BHT), potassium hydroxide (KOH), lithium perchlorate, and β-glucuronidase (type H-1, contains minimum 300,000 U/g β-glucuronidase activity and minimum 10,000 U/g sulfatase activity) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Diethyl ether was obtained from Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA), and HPLC-grade methanol, ethanol, hexane, and glacial acetic acid were from Fisher (Fair Lawn, NJ, USA).

Subjects

Healthy participants (5 women, 5 men) were recruited and written informed consent was obtained prior to inclusion in the trial. Participants were not smokers, did not take any dietary supplements at least 3 weeks prior to and during the study, and restricted their physical activity to <5 h/wk. Routine blood serum chemistry assays were performed at Good Samaritan Hospital (Corvallis, OR, USA) and were within the normal limits for all subjects. For subject characteristics see Table 1. The study protocol was approved by the Institutional Review Board at Oregon State University.

Table 1.

Subject characteristics and blood lipids at screening.

	Women (n=5)	Men (n=5)
Age (y)	25.4 ± 1.8	30.6 ± 2.6
BMI (kg/m2)	23.7 ± 0.6	24.9 ± 1.0
Plasma lipids (mmol/L)
Total cholesterol	4.40 ± 0.22*	4.97 ± 0.11
LDL cholesterol	2.40 ± 0.17#	3.14 ± 0.13
VLDL cholesterol	0.33 ± 0.06	0.52 ± 0.08
HDL	1.67 ± 0.16	1.31 ± 0.11
Triacylglycerols	0.72 ± 0.15	1.12 ± 0.18
Total Lipids	5.1 ± 0.2*	6.1 ± 0.3
Plasma tocopherols
α-Tocopherol (μmol/L)	19.4 ± 3.4	18.3 ± 2.4
γ-Tocopherol (μmol/L)	1.8 ± 0.5*	3.5 ± 1.5
α-Tocopherol per lipids(mmol/mol)	3.8 ± 0.3	3.0 ± 0.2
γ-Tocopherol per lipids(mmol/mol)	0.34 ± 0.4	0.57 ± 0.10

Shown are the baseline serum lipids, plasma tocopherols, and the tocopherol/lipid ratios (mean ± SEM). Lipids are defined as the sum of the serum total cholesterol and triglycerides, expressed as mmol/L. Statistical differences between values of women and men were calculated by an unpaired Student's t-test; significantly different at:

^*P<0.05,

^#P<0.01.

Experimental diets and study design

Corn and sesame oils used for the preparation of the muffins were equalized with respect to concentrations of α-T (17.9 mg/100 g corn oil; 15.6 mg/100 g sesame oil) and of γ-T (84.9 mg/100 g corn oil; 84.0 mg/100 g sesame oil). The two muffins made with either oil contained approximately 13.0 mg α-T and 2.5 mg γ-T. The two sesame oil muffins provided 135.4 mg sesame lignans (sesamin 93.8 mg; sesamolin, 41.6 mg). Sesame oil or corn oil (with equalized vitamin E concentrations in each oil) was used to prepare muffins from 650 g wheat flour, 250 g sugar, 25 g baking powder, 10 g salt, 130 g whole egg, 440 g milk, and 275 g oil by mixing all ingredients and weighing 50 g of the resulting batter into paper baking cups. Muffins were baked for 25 min at 205°C and stored at −20°C until used in the trials.

Subjects were randomly assigned to one of two treatments in a crossover design with a four-week washout period between treatments. A standard dinner was provided at 1800 on the evening before the trial and subjects were instructed to abstain from all food and beverages with the exception of water overnight. In the morning, blood samples were drawn from the antecubital arm vein into tubes containing 0.05 mL 15% K₃ EDTA (Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA). Subsequently, a standardized breakfast was served, including two sesame oil or corn oil (control) muffins and a capsule containing ~50 mg each deuterated d₆-α- and d₂-γ-tocopheryl acetates.

Blood samples were drawn 3, 6, 9, 12, 24, 36, 48, and 72 h after treatment. Meals were standardized during the period of 12 h before to 12 h after the dietary intervention and the subjects recorded their food consumption during the 2 consecutive days. For separation of blood plasma, samples were centrifuged (500 × g, 15 min, 4 °C), the plasma snap frozen in liquid nitrogen and stored at −80°C until analyzed. Urine was collected from 1 h prior to treatment on the first day of the trial; the urine bottles were exchanged at 0, 6, 12, 24, 36, 48, and 72 h after treatment. Urine volumes were recorded and 15 mL aliquots for each period were stored at −20°C until analyzed. After a 4-week washout period, treatments (sesame or corn oil muffins) were switched and the above procedures repeated.

Quantification of plasma labeled and unlabeled tocopherols

Tocopherols were extracted from plasma according to the method of Podda et al. (31). Briefly, plasma was saponified with saturated alcoholic KOH and the tocopherols extracted with hexane. An appropriate aliquot was dried under N₂-gas, the residue re-suspended in MeOH:EtOH (1:1, v:v) containing d₉-α-T as internal standard and injected into the LC system. For LC-MS analysis of labeled and unlabeled tocopherols, a Waters (Milford, MA, USA) 2690 Separations Module equipped with a Symmetry LC-18 column (Waters; 4.6 × 75 mm, 3.5 μm particle size; mobile phase, 100% methanol; flow rate, 1 mL/min) and a negative atmospheric pressure chemical ionization probe was used as previously described (32).

Quantification of plasma and urine labeled and unlabeled CEHC metabolites

Plasma and urinary CEHCs were extracted using a modified method of Lodge et al. (33). In brief, a known amount of trolox (internal standard) was added to either 0.5 mL plasma or 1 mL urine, before incubation for 30 min at 37°C with 100 μL β-glucuronidase (10 mg/mL dissolved in 0.01 M potassium phosphate buffer, pH 6.8), and 20 μL of H₂O containing 1% ascorbic acid. After incubation, the samples were acidified by the addition of 10 μL 12 M HCl. CEHCs were extracted with 5 mL of diethyl ether, dried under nitrogen gas and re-suspended in H₂O:MeOH (1:1, v:v), containing 0.1% (v:v) acetic acid, and 10 μL injected into the LC system described above equipped with a SymmetryShield™ RP-18 column (Waters; 3.0 × 150 mm, 3.5 μm particle size) and the solvent was delivered by a modified gradient method of Himmelfarb et al. (34). The system was first equilibrated with 50:50 H₂O:MeOH (both containing 0.1% acetic acid) for 1 min, followed by a linear gradient to 80% MeOH at 6 min at a flow rate of 0.25 mL/min. These conditions were maintained for 15 min, followed by a 5 min wash period with 95% MeOH, at which time original conditions were returned to and run for 5 min prior to injection of the proceeding sample. Samples were detected using a Micromass (Manchester, UK) ZQ 2000 single-quadrupole mass spectrometer with an electrospray ionization source (capillary voltage, 2.5 V; sample cone voltage, −30 V; desolvation temperature, 150°C; desolvation gas (nitrogen) flow, 160 L/h; nebulizer gas (nitrogen) pressure, 80 psi; cone gas (nitrogen) flow, 50 L/h), dwell time per compound, 0.20 s. Single ion recording mass-to-charge (m/z) ratios for molecular ions were as follows: d0-α-CEHC, m/z 277; d6-α-CEHC, m/z 283; d0-γ-CEHC, m/z 263; d2-γ-CEHC, m/z 265, and trolox, m/z 249. Sample CEHC concentrations were calculated from the ratio of the peak area of the corresponding ion to that of the internal standard trolox peak; deuterated CEHCs were calculated using the corresponding non-deuterated CEHC standards.

Quantification of plasma triacylglycerols and cholesterol and urinary creatinine

Plasma triacylglycerols and cholesterol were determined using the respective ThermoDMA Kits (Louisville, CO, USA). Urinary creatinine was quantified spectrophotometrically at a wavelength of 500 nm after reaction with picric acid according to the Jaffé reaction (35).

Statistical analyses

The maximal concentrations (C_max) and the time to reach maximal concentrations (T_max) were identified by visual inspection of each individual's plasma concentration data. Areas under the curve (AUC) were calculated using the trapezoidal rule. Fractional disappearance rates (FDRs) of d₆-α- and d₂-γ-tocopherols and d₆-α- and d₂-γ-CEHCs were calculated separately for each individual as described previously (36). Repeated measures MANOVA was performed using JMP Statistical Discovery Software (SAS Institute, Cary, NC) to evaluate effects attributed to oil type and to gender. When significant interactions were found then an unpaired Student's t-test for between gender comparisons or a one-tailed, paired Student's t-test for comparisons of the two treatments within the same subjects was carried out. Differences were considered significant at a p<0.05 level. Data are expressed as means ± SEM unless otherwise noted.

RESULTS

Participant Characteristics

All participants had normal blood levels of analytes measured in routine chemistry panels (data not shown) and were within the normal range for BMI and blood lipids (Table 1). Men, as compared with women, had significantly higher plasma γ-T concentrations, as well as serum total and LDL cholesterol at the time of screening; plasma α-T concentrations were similar in all subjects (Table 1). Neither plasma α-T nor γ-T concentrations were different between men and women, when expressed per serum total lipids (sum of cholesterol and triacylglycerols) because total lipid concentrations were lower in women than in men (p <0.05).

No significant differences in the dietary intakes of macro- and micronutrients, including vitamin E, were observed between treatment groups or between men and women throughout the trials (data not shown).

Plasma tocopherols and CEHCs

With respect to plasma d₆-α-T concentrations and calculated kinetic parameters, sesame oil compared with corn oil-containing muffins had no significant effects on any of these parameters (Figure 1A, Table 2). Specifically, plasma d₆-α-T AUC, C_max, and T_max values did not differ between the treatments (Table 2). Participants' plasma unlabeled (d₀)-α-T and d₀-γ-T concentrations were unresponsive to the type of muffin consumed (data not shown).

Figure 1. Plasma d₆-α and d₂-γ-tocopherol and d₂-γ-CEHC concentrations.

Plasma d₆-α-T (A), d₂-γ-T (B) and d₂-γ-CEHC (C) concentrations (μmol/L, mean + SEM) following ingestion of muffins prepared from unrefined sesame oil (squares, dotted lines) or corn oil (triangles, solid lines) simultaneously with a capsule containing 50 mg each d₆-α- and d₂-γ-TAcs in men (n= 5, filled symbols) and in women (n= 5, open symbols). Plasma samples were collected periodically for 72 h. By 72 h, the d₂-γ-T (B) concentrations were below levels of detection for many subjects, thus the means are not shown.

Table 2.

Kinetic parameters calculated from plasma d₆-α-T, d₂-γ-T and d₂-γ-CEHC concentrations in subjects consuming d₆-α-TAc and d₂-γ-TAc together with sesame oil or corn oil muffins, respectively.

	d6-α-Tocopherol					d2-γ-Tocopherol					d2-γ-CEHC
	corn oil	sesame oil	oil	gender	Oil × gender	corn oil	sesame oil	oil	gender	Oil × gender	corn oil	sesame oil	oil	gender	Oil × gender
AUC (μmol/L·h)
Women (n=5)	164 ± 19	162 ± 27	NS	NS	NS	20 ± 5	21 ± 3	NS	p<0.07	NS	4.8 ± 1.0	4.9 ± 0.8	p<0.05	NS	p<0.03
Men (n=5)	194 ± 24	193 ± 16				29 ± 3	34 ± 5				5.0 ± 1.0	2.6 ± 0.8*,+
Cmax (μmol/L)
Women (n=5)	4.2 ± 0.5	4.8 ± 0.4	NS	NS	NS	1.3 ± 0.3	1.3 ± 0.2	NS	p<0.05	NS	0.18 ± 0.05	0.29 ± 0.08	NS	NS	p<0.01
Men (n=5)	4.3 ± 0.6	4.3 ± 0.4				1.8 ± 0.3	2.0 ± 0.2				0.21 ± 0.04	0.10 ± 0.03*,+
Tmax (h)
Women (n=5)	16 ± 3	16 ± 3	NS	NS	NS	10 ± 2	16 ± 3	p<0.03	NS	NS	13 ± 3	19 ± 5	p<0.005	NS	NS
Men (n=5)	19 ± 3	17 ± 3				10 ± 1	11 ± 1				14 ± 2	22 ± 2
FDR (pools/day)
Women (n=5)	0.57 ± 0.01	0.53 ± 0.02	NS	P<0.002	NS	2.15 ± 0.14	1.94 ± 0.14	NS	NS	NS	1.38 ± 0.09	1.48 ± 0.08	NS	NS	NS
Men (n=5)	0.45 ± 0.01	0.42 ± 0.01				1.49 ± 0.04	1.46 ± 0.05				1.2 ± 0.0	1.0 ± 0.1

Shown are mean ± SEM. No d₆-α-CEHC was detected in plasma. Repeated measures MANOVA was performed using JMP Statistical Discovery Software (SAS Institute, Cary, NC) to evaluate effects attributed to oil type and to gender. Where there were significant interactions, post-hoc t-tests were performed;

^*P<0.05 for paired comparisons of corn vs sesame oil within each gender.

⁺P<0.05 for unpaired comparisons between men and women within each treatment group.

In contrast, consumption of sesame oil compared with corn oil muffins delayed the time of the peak (T_max) in the plasma kinetics of concurrently ingested d₂-γ-T in both men and women (Figure 1B, Table 2). The dietary treatments did not significantly alter any of the other parameters of d₂-γ-T kinetics (Table 2).

Differences between plasma α- and γ-T concentrations have been attributed in part to increased γ-T metabolism (13). Therefore, we sought to evaluate the efficacy with which sesame oil might alter vitamin E metabolism by measuring plasma CEHCs derived from the administered deuterated α- and γ-Ts. Similar to our previous studies, no d₆-α-CEHC was detected in plasma or urine (29), suggesting that the dose of d₆-α-T was insufficient to be metabolized and detectable as plasma d₆-α-CEHC.

In the present study, differences in d₂-γ-CEHC concentrations were observed with respect to the sesame oil treatment in men, but not in women (significant interactions between oil and gender, for C_max and AUC). Sesame oil consumption significantly (P<0.05) decreased both plasma C_max and d₂-γ-CEHC AUCs (Figure 1C,Table 2). Thus, sesame oil consumption in men not only delayed the T_max of plasma d₂-γ-T concentrations (discussed above), it also halved the maximum plasma d₂-γ-CEHC concentrations and the AUCs. Remarkably, sesame oil consumption also decreased (p<0.05) mean concentrations over 72 h of both plasma d₀-α- and d₀-γ-CEHCs in men, but not women (Figure 2).

Figure 2. Sesame oil consumption in men decreased plasma d₀-α and d₀-γ-CEHC concentrations.

Plasma d₀-α-CEHC (A) and d₀-γ-CEHC (B) concentrations (nmol/L, mean + SEM) from all samples from 3 to 72 h were averaged for each subject (n=5 men, 5 women) following ingestion of sesame oil (solid bar) or corn oil (hatched bar) muffins. Repeated measures MANOVA was performed using JMP Statistical Discovery Software (SAS Institute, Cary, NC) to evaluate effects attributed to oil type and to gender. Sesame oil consumption decreased plasma d₀-α-CEHC concentrations (interaction P < 0.01, only in men, P<0.05). There was a main effect of oil type on plasma d₀-γ-CEHC concentrations (P< 0.05).

Some notable differences in vitamin E kinetics were observed that were related only to gender differences. Women had significantly (p<0.002, main effect) greater fractional disappearance rates (FDR) of plasma d₆-α-T concentrations than did men (Figure 1A, Table 2). Similarly, when the d₂-γ-T FDRs were averaged for the two dietary treatments for each subject, women compared with men also had greater d₂-γ-T FDRs (2.0 ± 0.1 vs 1.5 ± 0.0 pools per day, p<0.05). When concentrations were adjusted for serum total lipids at each time point, and kinetics similarly calculated, women's plasma d₆-α-T/lipids FDRs were also greater than men's (main effect, p<0.01, not shown).

Urinary CEHC excretion

We observed that urinary d₂-γ-CEHC concentrations during the first 24 h of the study were significantly decreased by consumption of sesame oil muffins both in women and in men (Table 3). Since sesame lignans and their metabolites appear to be eliminated from the body and excreted in urine within 24 h following consumption by humans (37), any potential physiological effects would only be expected during this interval. Thus, it is not surprising that sesame oil consumption did not have effects in the subsequent urine collections (data not shown). No d₆-α-CEHC was detectable in the urine samples (data not shown).

Table 3.

Urinary excretion of d₀-α-CEHC, d₀-γ-CEHC and d₂-γ-CEHC (nmol/g creatinine) over 24 h in subjects consuming d₆-α-TAc and d₂-γ-TAc together with sesame oil or corn oil muffins, respectively.

	d0-α-CEHC					d0-γ-CEHC					d2-γ-CEHC
	corn oil	sesame oil	oil	gender	Oil × gender	corn oil	sesame oil	oil	gender	Oil × gender	corn oil	sesame oil	oil	gender	Oil × gender
Women (n=5)	0.87 ± 0.15	0.92 ± 0.44	NS	P<0.008	NS	2.00 ± 0.26	1.83 ± 0.60	NS	P<0.008	NS	1.15 ± 0.24	0.81 ± 0.31	P<0.030	P<0.055	NS
Men (n=5)	0.23 ± 0.04	0.11 ± 0.03				1.00 ± 0.21	0.59 ± 0.17				0.56 ± 0.16	0.27 ± 0.12

Shown are mean ± SEM. Repeated measures MANOVA was performed using JMP Statistical Discovery Software (SAS Institute, Cary, NC) to evaluate effects attributed to oil type and to gender.

Both d₀-α- and d₀-γ-CEHC urinary excretion were higher in women than in men, but sesame oil consumption had no significant effect on their excretion.

DISCUSSION

This study was designed to test the hypothesis that humans consuming sesame lignans (sesamin and sesamolin) from unrefined sesame oil would have increased plasma γ-T concentrations likely as a result of inhibiting the metabolism of γ-T to γ-CEHC. These expectations are in accordance with previously published findings from human (27,28) and rat (24,25,38,39) studies that showed markedly increased plasma γ-T concentrations after dietary intervention with sesame seeds, sesame oil, or isolated sesame lignans. Although sesame oil consumption delayed the peak in d₂-γ-T concentrations in both men and women (Table 2), none of the other d₂-γ-T kinetic parameters were altered by sesame oil. However, we found that plasma d₂-γ-CEHC AUCs, and C_maxs were significantly lower in men after consuming sesame oil muffins compared with corn oil muffins (Figure 1C, Table 2), suggesting that sesame lignans interfered with d₂-γ-T metabolism. Surprisingly, similar decreases in response to sesame oil consumption were not found for women's plasma d₂-γ-CEHC concentrations, although sesame delayed the peak in the d₂-γ-CEHC concentrations in both men and women.

In conjunction with the significant decrease in the plasma d₂-γ-CEHC AUCs, and C_maxs, a reduction in urinary d₂-γ-CEHC excretion was observed in both genders during the first 24 h of the sesame oil trial. It is likely that women's vitamin E metabolism was affected by sesame lignans, as observed previously (27), but did not result in differences in their plasma d₂-γ-CEHC concentrations. This speculation is supported by the findings that overall urinary excretion of unlabeled α- and γ-CEHC and d₂-γ-CEHC was higher in women than in men (Table 3).

The percentage of d₂-γ-T dose recovered over 72 h as urinary d₂-γ-CEHC in men was nearly doubled during the corn oil (2.1% ± 0.6%) intervention compared with the sesame intervention (1.2% ± 0.4%), but these differences did not reach statistical significance; in women the overall percentage of dose excreted was 2.5% ± 0.7% and 2.3% ± 0.5%, during the corn and sesame oil trials, respectively. Parker and co-workers (26) previously demonstrated in cells in culture that the sesame lignan, sesamin, at very low concentrations (1 μM) almost completely inhibited the formation of the side-chain truncated metabolite γ-CEHC from γ-T. They proposed the involvement of cytochrome P450 (CYP) enzymes in the initial ω-hydroxylation of the terminal methyl group of the tocopherol side-chain (18) and its inhibition by sesamin (26). These findings offer a ready explanation for the pronounced decrease in plasma γ-CEHCs concentrations due to sesame lignan consumption in our study. This mechanism is further supported by reports of a reduced excretion of γ-CEHC in the urine of rats fed sesame seeds, isolated sesame lignans (sesamin or sesaminol), or ketoconazole (21). The data presented herein, however, shows in humans that γ-T metabolism can be inhibited by the simultaneous consumption of γ-T and sesame lignans. It is remarkable, that this γ-T-sparing activity was observed after the consumption of only a single oral dose of 94 mg sesamin and 42 mg sesamolin. Repeated ingestion of sesame lignans results in an even more pronounced increase in γ-T concentrations, as observed by Cooney et al. (28) in subjects consuming muffins prepared with ground sesame seeds on three consecutive days.

The potent inhibition of CYP-mediated vitamin E metabolism by sesame lignans is of particular importance with regard to clinical nutrition. Chemicals with a methylenedioxyphenyl function, such as sesamin and sesamolin, are known to form complexes with CYPs, thereby irreversibly inactivating the enzymes (40). CYPs are centrally involved in the detoxification of xenobiotics, including many pharmaceutical agents. CYP3A4, for example, metabolizes more than 50% of prescription drugs (41). Thus, simultaneously ingested sesame lignans may alter the bioavailability and biopotency of drugs by altering their in vivo-conversion to the bioactive forms or by slowing down their elimination from the body. Furthermore, phase I enzymes, such as CYPs, are involved in the activation, as well as the elimination of pro-carcinogens; thus, sesame lignans may hypothetically interfere with cancer development. In female rats, feeding of a mixture of sesamin and episesamin at 0.2% (by weight) in the diet, significantly reduced the formation of chemically-induced mammary carcinomas (42).

Throughout the current investigation, we observed considerable differences in the handling, metabolism, and excretion of tocopherols and their water-soluble metabolites between men and women. The faster plasma disappearance of both plasma d₆-α-T and d₂-γ-T in women compared with men (Table 2) and increased urinary excretion of γ-CEHC relative to α-CEHC is consistent with our previous results (29). In general, the production of α-CEHC from α-tocopherol is limited (17,18). Our previous study examining the relative metabolism of α-T and γ-T showed in normal individuals that plasma α-CEHC concentrations are 1/10^th of those of γ-CEHC (29). Additionally, Shultz et al. (43) showed that α-tocopherol intakes in human had to be over 150 mg/day in order to detect plasma α-CEHC. We therefore did not find it surprising that d₆-α-CEHC was not detectable with a single dose of 50 mg/day.

These data suggest that further studies comparing vitamin E metabolism in men and women are warranted.