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-Tocopherol Biokinetics1,2
Centre for Nutrition and Food Safety, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK
3To whom correspondence should be addressed. E-mail: j.lodge{at}surrey.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol (-T) in blood components was investigated in normolipidemic (N; total cholesterol < 5.5 mmol/L and triglycerides < 1.5 mmol/L, n = 9), hypercholesterolemic (HC; total cholesterol > 6.5 mmol/L and triglycerides < 1.5 mmol/L, n = 10), and combined hypercholesterolemic and hypertriglyceridemic (HCT; total cholesterol > 6.5 mmol/L and triglycerides > 2.5 mmol/L, n = 6) subjects. Subjects ingested 150 mg hexadeuterated RRR--tocopheryl acetate, and blood was collected up to 48 h after ingestion. Labeled -T was measured in plasma, lipoproteins, erythrocytes, platelets, and lymphocytes by liquid chromatography/mass spectroscopy. In plasma, HC had an earlier time of maximum concentration (6 h) compared with N and HCT (12 h) (P < 0.05). HCT had a lower uptake of labeled -T (P < 0.005) and a longer half-life (P < 0.05). In chylomicrons, the maximum labeled -T concentration was higher in HC compared with N and HCT (P < 0.00005); however, HCT had a lower uptake of labeled -T in LDL. In all groups, the lowest density LDL subfraction contained more labeled -T than denser subfractions (P < 0.05). In platelets, lymphocytes, and erythrocytes, the areas under the labeled -T concentration vs. time curves were in the order N > HC > HCT. In lymphocytes, differences in labeled -T were found at 6 and 48 h (P < 0.05). These data demonstrate that there are differences in the uptake of newly absorbed -T into blood components in hyperlipidemia. Because these blood components are functionally affected by vitamin E, reduced uptake of -T may be relevant to the pathogenesis of atherosclerosis.
KEY WORDS: • deuterated • tocopherol • biokinetics • blood cells • hyperlipidemia
Vitamin E is a group of 8 compounds (, 
, ß, and 
tocopherols and tocotrienols), with -tocopherol (-T)4 being the predominant form in the body, comprising over 90% of vitamin E (1,2). Epidemiology studies have consistently demonstrated a relation between both vitamin E intakes (3–5) and steady-state plasma vitamin E concentrations (6,7), with the risk of coronary heart disease (CHD). The hyperlipidemias are independent risk factors for heart disease (8). High plasma levels of both cholesterol (9) and triglycerides (10) have been linked to increased mortality from CHD. Hyperlipidemia is associated with the atherogenic lipoprotein phenotype (11,12) (characterized by a preponderance of smaller dense LDL particles) and impaired platelet (13,14) and lymphocyte (15,16) function. These blood components are functionally influenced by -T treatment (17–19). Indeed, many of the critical cells associated with atherosclerotic lesion development are influenced by -T via a variety of antioxidant and nonantioxidant mechanisms (20,21). Such studies provide a rational for the use of vitamin E as a cardioprotective agent and suggest that an adequate vitamin E status is important for decreasing CHD risk.
Vitamin E homeostasis in hyperlipidemia is not well documented. Vitamin E is transported within lipoproteins while circulating in the blood. As a consequence, vitamin E concentration is closely correlated with that of cholesterol and total lipid (22,23); hence, hypercholesterolemics usually have increased concentrations of plasma vitamin E (uncorrected for cholesterol concentrations) compared with normolipidemics (24,25), whereas decreased concentrations have been observed in hypocholesterolemia (26). Similarly, hypertriglyceridemics also appear to have higher plasma vitamin E (27).
Erythrocyte vitamin E concentrations were found to be lower in hypercholesterolemics, even when plasma levels were similar (24,28), showing the limitations of measuring plasma levels alone. In addition, we recently found reduced -T concentrations in the lymphocytes and platelets of smokers compared with nonsmokers, which suggests that lymphocyte and platelet -T content may be a potential marker of vitamin E status (29). These data suggest that in impaired metabolic states, including hyperlipidemia, the transfer of vitamin E from plasma lipoproteins to target cells may be impaired. Because vitamin E distribution is related to the kinetics of lipoprotein metabolism, abnormalities of the plasma lipid status associated with hyperlipidemia may affect functional vitamin E status. This may have implications related to the activity of those cells that are responsive toward vitamin E.
With this in mind, we have investigated vitamin E biokinetics in plasma, lipoproteins, erythrocytes, platelets, and lymphocytes in normolipidemic, hypercholesterolemic, and combined hypercholesterolemic and hypertriglyceridemic individuals.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-5, 7-(CD3)2-tocopheryl acetate and all rac--5 (CD3)-tocopheryl acetate were kind gifts from Cognis Nutrition and Health. Purities of the acetates was 98.8% for both species. Isotopic purity was determined to be >99.9% by LC/MS. Hexadeuterated RRR--tocopheryl acetate was encapsulated into hard gelatin capsules (150 mg) for human consumption. Trideuterated all rac--T was used as an internal standard. Reagents and solvents. LC/MS grade methanol (LC-MS Chromasolv), butylated hydroxytoluene, Histopaque-1077, and lithium perchlorate were purchased from Sigma-Aldrich Chemical. Sodium dodecyl sulfate was from BDH, hexane (HPLC grade) was from Fisher, absolute ethanol was from Hayman, and phosphate-buffered saline tablets were from Oxoid.
Study protocol. Normolipidemic and hyperlipidemic subjects were recruited from local lipid clinics associated with the Royal Surrey County Hospital and from the general population by advertisements in local newspapers. Selection criteria stated subjects must be nonsmoking, not be taking dietary supplements, be without gastrointestinal or hepatic conditions, and not be taking lipid-lowering drugs. We recruited subjects based on the following lipid chemistry: normolipidemics (N group) total plasma cholesterol < 5.5 mmol/L, LDL cholesterol < 3.5 mmol/L, and plasma triglycerides < 1.5 mmol/L; hypercholesterolemics (HC group) total plasma cholesterol > 6 mmol/L, LDL cholesterol > 3.5 mmol/L, and plasma triglycerides < 1.5 mmol/L; combined hyperlipidemics (HCT group) total plasma cholesterol > 6 mmol/L, LDL cholesterol > 3.5 mmol/L, and plasma triglycerides > 1.7 mmol/L.
Volunteers were given 150 mg encapsulated hexadeuterated RRR--tocopheryl acetate with a standard breakfast containing 40 g fat. Blood samples were taken 3, 6, 9, 12, 24, and 48 h after ingestion of the capsule. Blood components were immediately isolated from the whole blood. Volunteers consumed a standard lunch 4.5 h after the breakfast, which contained 2.5 g fat, and, after 8 h, food and beverage consumption was no longer controlled.
The study was approved by the University of Surrey Advisory Committee on Ethics and by the South West Surrey Local Research Ethics Committee.
Sample isolation and analysis. Erythrocytes, platelets, and lymphocytes were isolated from fresh whole blood by standard methods as described (29,30). Plasma was collected during platelet isolation (platelet-poor plasma). All samples were equally divided, frozen in liquid nitrogen, and stored at –80°C prior to analysis.
Chylomicrons were isolated from previously frozen plasma as described (31). VLDL, LDL, and HDL were then extracted from this chylomicron-free plasma by sequential ultracentrifugation through a discontinuous KBr gradient, as described (32). Freezing has been shown not to influence -T levels either from plasma or from isolated lipoproteins (33). A Beckman Coulter 70.1 Ti rotor and Beckman Optima XL-100 ultracentrifuge were used for all lipoprotein isolation. LDL subclasses were isolated from fresh plasma by ultracentrifugation, using a self-forming density gradient of iodixanol as described (34). Aliquots of isolated lipoproteins were immediately analyzed for cholesterol, triglycerides, protein, and -T, using methods described below.
Due to difficulties obtaining enough blood for lipoprotein isolation from certain subjects, lipoproteins were isolated from 6 of 9 (N), 5 of 10 (HC), and 5 of 6 (HCT) subjects.
Vitamin E extraction and analysis. Total vitamin E was extracted from all samples as described (35). Tocopherols were analyzed by LC/MS, using a method we recently developed (36). The system used was a Micromass LCT (Waters), which combines a Waters Alliance System comprising a solvent delivery system, online degasser, peltier-cooled autosampler (set at 4°C), controller, and column oven (set at 25°C), in conjunction with a Time-of-Flight Mass Spectrometer. Tocopherols were separated on a Waters SymmetryTM Column (2.1 x 50 mm, C18, 3.5 µm) with a mobile phase consisting of 100% methanol (LC/MS Chromasolv, Sigma-Aldrich).
Biochemical analysis. Total cholesterol and triglycerides were measured in plasma and lipoprotein fractions, using enzymatic kits supplied by Randox, and were analyzed automatically using a SPACE biochemical analyzer (Alfa-Wasserman). Total protein content of lipoproteins, platelets, and lymphocytes were determined using a colorimetric protein kit based on the Bradford assay (BioRad).
Data analysis and statistics.
Noncompartmental kinetic parameters associated with plasma labeled -T concentration vs. time profiles were determined using the PK Solutions Version 2.0 software (Summit Research Services). Data are expressed as means with SEM. Data were found to be normally distributed using the Kolmogorov-Smirnov test, and therefore parametric tests were used. Two-way repeated measures ANOVA, with group (N, HC, HCT) as a between-subject factor and time (3 to 48 h) as a within-subject factor, was used to test for group effects and group x time interactions. Differences in kinetic parameters between groups were tested using one-way ANOVA. Tukey’s honestly significant difference post-hoc test was used when a significant result was found. Results were considered significant at the 95% confidence level (P < 0.05). The software package Statistica for Windows (Statsoft) was used for all statistical analysis.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-T concentrations in plasma.
Baseline unlabeled (preexisting) plasma -T concentrations, uncorrected for cholesterol, were: N, 25.20 ± 7.6; HC, 38.88 ± 11.37; and HCT, 33.19 ± 1.47 µmol/L. The HC group had a higher unlabeled -T concentration than both the N and HCT groups (P < 0.05).
After ingestion of 150 mg deuterated RRR--tocopheryl acetate, there was a decrease in the plasma concentration of unlabeled -T over time (P < 0.0001) in all groups (Fig. 1A).Plasma labeled -T increased in concentration (P < 0.0001) with time in all groups to a maximum between 6 and 9 h in the HC group and 12 h in the N and HCT groups (Fig.1B). There was a significant group x time interaction in plasma labeled -T concentration (P < 0.0005) due to reduced labeled -T concentrations at 6 and 9 h in the HCT group. Considerable interindividual variation was observed in maximum plasma labeled -T concentrations (Fig. 2), but individual subjects within each group displayed a similar profile.
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-T response was subjected to kinetic analysis (Table 2). HCT showed a longer half-life (P < 0.05) and a larger volume of distribution (P < 0.05) than the N and HC groups. The HC group had a shorter time of maximum concentration than the N and HCT groups (P < 0.01).
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-T concentrations in lipoproteins.
After ingestion of 150 mg deuterated RRR--tocopheryl acetate, labeled -T increased in lipoproteins over time (P < 0.0001) (Fig. 3). In chylomicrons (Fig. 3A), labeled -T increased sharply, up to a maximum at 6 h, then decreased. There was a significant group x time interaction for labeled -T concentrations (P < 0.00005), due to the -T concentration in HC at 6 h being 
300% higher than that in N and HCT. The areas under the labeled -T concentration vs. time curves (AUC; µmol · h/L) in chylomicrons were 177 ± 55 (N), 291 ± 52 (HC), and 176 ± 23 (HCT). In VLDL (Fig. 3B), time of maximum concentration occurred between 9 and 12 h in all groups, and AUCs were 1050 ± 76 (N), 656 ± 206 (HC), and 832 ± 63 (HCT). In LDL (Fig. 3C), maximum concentration occurred between 12 and 24 h in all groups with AUCs of 534 ± 128 (N), 515 ± 36 (HC), and 358 ± 18 (HCT). In HDL, the labeled -T concentration vs. time profiles in HDL were almost identical among the groups (data not shown), with AUCs of 48 ± 5 µmol · h/L in all groups.
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-T in LDL subclasses (density range 1018–1043 g/L) at 12 and 24 h after ingestion of 150 mg deuterated RRR--tocopheryl acetate were analyzed. Labeled -T concentrations (µmol/mmol cholesterol) in selected subfractions were: 1018 g/L, 3.23 ± 0.3 (N), 2.55 ± 0.4 (HC), and 2.79 ± 0.5 (HCT); 1022 g/L, 2.14 ± 0.3 (N), 1.98 ± 0.2 (HC), and 1.94 ± 0.3 (HCT); 1043 g/L, 1.44 ± 0.2 (N), 1.76 ± 0.2 (HC), and 1.63 ± 0.1 (HCT). There was a higher labeled -T concentration in the 1018 g/L subfraction than in the denser (
1022 g/L) subfractions (P < 0.05) but no differences among groups. There were no differences between the 12 and 24 h samples.
Labeled -T concentrations in blood components.
After ingestion of 150 mg deuterated RRR--tocopheryl acetate, labeled -T concentration was measured in erythrocytes, platelets, and lymphocytes (Fig. 4). In erythrocytes (Fig. 4A), the concentration of labeled -T in all groups showed a gradual increase in concentration up to a maximum at 24 h. The AUCs (µmol · h/L) were 174 ± 40 (N), 165 ± 59 (HC), and 130 ± 43 (HCT). In platelets (Fig. 4B), labeled -T concentration showed a biphasic response characterized by an initial peak at 6 h, decreasing by 9 h, followed by a more gradual increase in concentration up to 48 h. The AUCs (µmol · h/L) were 18 ± 4 (N), 15 ± 5 (HC), and 13 ± 5 (HCT). Lymphocytes (Fig. 4C) also showed a gradual increase in labeled -T concentration, which was still increasing after 48 h. There was little increase in labeled -T concentrations until 9 h in the N and HCT groups; however, in the HC group, there was an initial peak at 6 h, which was higher than the N and HCT groups (P < 0.05). At 48 h, labeled -T concentration was greater in N compared with HC and HCT (P < 0.05). The AUCs (µmol · h/L) were 12 ± 4 (N), 9 ± 4 (HC), and 8 ± 3 (HCT). In all blood components, the highest labeled -T concentrations were found in the N group, while the lowest labeled -T concentration at each time point was consistently found in the HCT group.
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-T biokinetics in blood components of subjects classified as normolipidemic (N), hypercholesterolemic (HC), or combined hypercholesterolemic and hypertriglyceridemic (HCT). Using this approach, the uptake of newly absorbed vitamin E can be directly monitored. We show here that hyperlipidemia is associated with reduced uptake of newly absorbed vitamin E into the plasma and into erythrocytes, platelets, and lymphocytes.
The plasma uptake profiles of labeled -T were different among the groups (Fig. 1). The N group displayed a maximum plasma concentration at 12 h, similar to that previously observed (36–38), whereas in the HC group, the time of maximum concentrations was significantly shorter at 6 h (P < 0.001). The HCT group displayed a different profile, with a lower uptake of newly absorbed (labeled) -T (P < 0.0005). Kinetic analysis (Table 2) demonstrated a reduced area under the labeled -T concentration vs. time curve (AUC), a significantly longer half-life (P < 0.05), and a greater volume of distribution (P < 0.05) in the HCT group. A larger volume of distribution implies that more of the -T is present in tissues compared with blood. The groups did not differ in their plasma profiles of preexisting -T. All groups had a decrease in unlabeled -T that coincided with the increase in labeled -T. This is a well established phenomenon, in which the hepatic -T transfer protein constantly replaces the preexisting -T in circulation with newly absorbed -T (37) and in this way regulates plasma -T concentrations. Large interindividual variation in the plasma responses of labeled -T was found in the present study (Fig. 2), consistent with similar studies (38). Although there was considerable heterogeneity in the maximum -T concentration, each group was associated with a distinctive -T concentration vs. time profile (described above), and all the subjects within that particular group had essentially the same profile. This suggests that, although the amount absorbed may differ, the pattern of plasma appearance depends on other factors (e.g., the plasma lipid status in the present study).
The analysis of labeled -T in isolated lipoproteins provided further insights into the differences in -T uptake in hyperlipidemic subjects. In chylomicrons, HC subjects had higher maximum -T concentrations compared with the N and HCT groups (P < 0.00005). The HC group also had the largest increase in triglyceride concentration at this time point (data not shown), suggesting that HC subjects produce larger chylomicron particles, containing large amounts of triglycerides and labeled -T. There is little in the literature that directly addresses the differences in chylomicron triglyceride secretion postprandially in HC and HCT individuals, and this merits further investigation. Combined hyperlipidemics (HCT) are characterized by a delayed clearance of plasma triglycerides, and a high fasting triglyceride level may have inhibited the incorporation of newly absorbed triglyceride (together with labeled -T) into chylomicrons, in favor of storage in the enterocyte pool.
Although HC subjects secreted chylomicrons with almost 300% more -T per particle, no differences in -T concentrations among groups were found in VLDL. However, in LDL, as in plasma, the HCT group had lower -T concentrations at each time point. LDL is a product of lipoprotein lipase-mediated VLDL hydrolysis, and, during this process, -T can be distributed to tissues (39). This suggests that in the present study, more -T is distributed in HCT subjects than in N or HC subjects. Hypertriglyceridemia is associated with reduced activity of lipoprotein lipase, and subjects with genetically impaired lipoprotein lipase activity retain newly absorbed -T in their triglyceride-rich lipoproteins (40), which become the major carriers of vitamin E in the absence of LDL. However, in the present study, -T was not retained in chylomicrons or VLDL in the HCT group. Thus, it appears that differences in the present study may be related to the transfer of -T to/from LDL.
The analysis of -T distribution among LDL subfractions is of interest, given that a preponderance of small dense LDL is a risk factor for CHD (8,11). It has previously been demonstrated that small, dense LDL contains less -T per particle than larger, less dense, particles (41,42), rendering these particles more susceptible to oxidation (43). In the present study, we found more newly absorbed -T in larger, less dense subfractions than in smaller, dense LDL (P < 0.05). This indicates that differences exist in the transfer of -T such that less dense LDL fractions tend to retain their -T, whereas more dense LDL can readily donate -T.
Hyperlipidemic subjects have a reduced uptake of newly absorbed -T into erythrocytes, platelets, and lymphocytes. Erythrocyte-labeled -T concentrations peaked at 24 h then started to decline at 48 h in all subjects. Erythrocytes are thought to obtain their vitamin E via tocopherol-binding proteins (44) and transfer from HDL has been suggested (45). Previous studies have reported lower steady-state vitamin E levels in erythrocytes of hyperlipidemics compared with normolipidemic controls (24) but no difference in the transfer of -T from donor HDL to erythrocytes in hyperlipidemic and normolipidemic subjects (45). -T uptake into lymphocytes was gradual, appearing after 6 h and continuing to increase up to 48 h in all subjects. Lymphocytes contain LDL receptors, which are potential mechanisms of vitamin E delivery (46). Some HC subjects have reduced binding activity of LDL receptors (47), which would influence vitamin E uptake. A distinct biphasic uptake of labeled -T into platelets was observed in all groups. The initial peak at 6 h corresponded to the time of maximum -T concentration in chylomicrons, indicating that during chylomicron hydrolysis there is transfer of -T to platelets. This effect was most pronounced in HC subjects, and these subjects had a 300% higher maximum -T concentration in chylomicrons. However, during the second "phase" of -T uptake (after 9 h), there was less -T taken up by the HC and HCT groups compared with the N group. Thus, it appears that independent mechanisms of uptake exist. Although platelets do not contain LDL receptors, the apoB moiety of LDL has recently been shown to interact with an unidentified receptor on the platelet membrane, which allows for the transfer of lipids (48). The biphasic response was also observed in the lymphocytes of HC subjects only, implying that this initial transfer of -T is a function of the chylomicron tocopherol concentration. It is interesting, however, that no such response was observed in the erythrocytes of HC subjects.
Hyperlipidemia is associated with greater platelet-endothelial adhesion (13) and increased platelet activation (14). Hyperlipidemia is also associated with impaired lymphocyte function (15,16). Both platelets and lymphocytes are functionally responsive toward -T treatment, as -T has been shown to modulate platelet adhesion and aggregation in supplementation studies and ex vivo experiments (17), and to enhance lymphocyte differentiation and proliferation in vitro and ex vivo (18). The impaired uptake of -T into platelets and lymphocytes in hyperlipidemic subjects could contribute to the altered platelet and lymphocyte function commonly observed in hyperlipidemia; however, further work is necessary to investigate any link between platelet and lymphocyte function, and vitamin E status within these components.
The findings in this study of a reduced uptake of newly absorbed -T into several blood components of hyperlipidemic subjects provides a useful insight into the possibility that -T regulation is different in hyperlipidemic subjects and that the delivery of -T to peripheral tissues may also be reduced. Because vitamin E is commonly seen as being cardioprotective through a combination of its antioxidant and nonantioxidant effects (20,21), an impairment of vitamin E status in hyperlipidemia could potentially influence CHD risk.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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-tocopherol in plasma, erythrocytes, platelets and lymphocytes in individuals with normal or raised plasma lipids], and the New York Academy of Sciences Vitamin E and Health Conference, May 24–26, 2004, Boston, MA [Lodge, J. K., Hall, W. L., Jeanes, Y. M. & Proteggente, A. R. Physiological factors influencing vitamin E biokinetics]. 
2 Financial support from the British Heart Foundation, The Royal Society, and the Medical Research Council (Y.M.J.).
4 Abbreviations used: -T, -tocopherol; AUC, area under the concentration versus time curve; CHD, coronary heart disease; HC, hypercholesterolemics; HCT, combined hypercholesterolemics and hypertriglyceridemics; N, normolipidemics.
Manuscript received 13 August 2004. Initial review completed 27 September 2004. Revision accepted 13 October 2004.
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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