2.1. Human studies
Aging is associated with enhanced oxidative stress and a decline in immune function. These changes may lead to an increase in the incidence and/or severity of microbial infections, autoimmune disorders, and degenerative diseases associated with chronic inflammation such as atherosclerosis, cancer, or neurodegenerative diseases such as Alzheimer’s disease (Fulop et al. 2006
). A marker of declined immune function in elderly is the decrease of IL-2, a cytokine important for the clonal expansion of T cells. A reduction in IL-2 levels leads to a decrease in clonal T cell expansion and thus to a decline in the specific immune response. Several studies have shown that supplementation of healthy elderly people with αT improves the overall immune response, as evidenced by an increased (i.e., restored) delayed-type hypersensitivity (DTH) reaction to various antigens, in vitro
T cell proliferation, IL-2 production, and inhibition of PGE2
Effect of vitamin E supplementation on immune response and inflammation in humans
Thus, supplementation of healthy elderly people with a relatively high dose of 800 mg/d αT for 4 weeks resulted in a three-fold increase in serum and peripheral blood mononuclear cell αT concentrations and a three-fold decrease in serum γT concentration, while no changes were observed in the placebo group (Meydani et al. 1990
). The DTH reaction was significantly increased in the αT-treated group, compared to both the placebo-treated group or the study group at baseline, both regarding the cumulative score (total diameter of induration of all positive reactions) and the antigen score (number of positive responses). Also, concanavalin A (Con A)-stimulated IL-2 production by isolated monocytes was significantly enhanced in the αT-supplemented group compared to cells at baseline. Furthermore, levels of the potent pro-inflammatory lipid mediator PGE2
were significantly reduced in αT-supplemented phytohemagglutinin (PHA)-stimulated monocytes compared to placebo. This decrease in PGE2
production might be responsible for the restoration of IL-2 production, as the former has been shown to suppress lymphocyte proliferation and IL-2 production (Goodwin and Webb 1980
; Walker et al. 1983
In another randomized controlled trial, Meydani et al.
showed that the DTH response was significantly increased in healthy elderly people also by αT supplementation at a dose of 200 mg/d (Meydani et al. 1997
). Furthermore, αT supplementation at this dose significantly boosted antibody titers to hepatitis B and tetanus vaccination compared to placebo. Immunoglobulin, T and B cell levels, were unaffected, however, as were antibody titers to diphtheria and pneumococcal vaccination.
Pallast et al.
showed that 100 mg/d of αT for 24 weeks only partially restored the DTH response in elderly people (while they did not observe any effect at 50 mg/d). This partial restoration of the DTH response was accompanied by a trend toward higher IL-2 production in isolated PHA-stimulated peripheral blood mononuclear cells. Interestingly, IFN-γ production decreased and IL-4 production increased in the groups receiving αT compared to baseline (but not placebo) (Pallast et al. 1999
). In contrast, De Waart et al.
could not find any beneficial effects in elderly people supplemented with 100 mg/d αT for 3 months, neither on ConA- or PHA-induced lymphocyte proliferation nor on antibody titers against common antigens such as milk protein (De Waart et al. 1997
). It therefore appears that the immunostimulatory effect of αT occurs at supplementation doses >100 mg/d. It is noteworthy that at these levels, αT significantly depresses plasma/serum γT concentrations. Whether γT (either alone or in combination with αT) has a beneficial effect on the decline of immune function in elderly has not been investigated.
In a randomized controlled trial, Graat et al.
examined the effect of αT supplementation on acute respiratory tract infections in well-nourished non-institutionalized elderly people (Graat et al. 2002
). Participants were given four supplement regimens, i.e.
, either 200 mg of all rac
-αT acetate, a multivitamin-mineral preparation containing the recommended dietary allowance level for each component, combined all rac
-αT plus multivitamin-minerals, or placebo, for a median period of 441 days. In this cohort, αT administration prompted no favorable effects on the incidence or severity of acute respiratory tract infections (upper and lower combined). Co-supplementation with the multivitamin-mineral preparation did also not result in a beneficial effect. However, when the two αT supplemented groups were combined, αT administration led to a significant increase
in both the duration and severity of respiratory tract infections. It is possible, however, that this effect was confounded by a slightly higher number of active smokers in the αT-supplemented group. In another study in elderly nursing home residents, oral administration of 200 IU αT/d for 1 year showed no effect on incidence or duration for upper and lower respiratory tract infections. However, when supplemented with αT compared to placebo, there were significantly fewer people in the subgroup of subjects that reported at least one incidence of upper respiratory tract infection. Common colds also occurred less often in the αT supplemented group than in the placebo group (Meydani et al. 2004
). The effect of αT supplementation on the incidence of respiratory tract infections was also analyzed in the subgroup of male smokers that participated in the Alpha-Tocopherol Beta-Carotene Cancer Prevention Study (Hemila et al. 2002
). Supplementation with 50 mg/d of αT for 4 years slightly reduced the incidence of common colds only in people ≥65 years of age. Although αT supplementation has a clear immunostimulatory effect in elderly people at doses >100 mg/d, the impact on respiratory tract infections (in which the adaptive immune response plays an important role) is relatively moderate. Elderly people that have low vitamin E levels probably benefit most from supplementation with αT. Whether high doses of αT may in fact exacerbate immunodeficiency in already αT-proficient elderly people also requires further investigation.
The observation that asthma patients or subjects with other allergies have reduced levels of αT has prompted several studies to test the potential benefits of αT to the alleviation of allergic symptoms, in which Th2 cytokines such as IL-4 and IL-5 are thought to play a major role. However, supplementation of 112 patients with documented hay fever with 800 mg/day αT for 2.5 months failed to decrease the percentage of days with serious symptom or medication use during pollen season compared to placebo control; only a small effect on certain nasal symptoms was noted (Shahar et al. 2004
). In another double-blind, placebo-controlled trial, supplementation of allergic rhinitis patients with 400 IU αT/day for four weeks also had no effect on symptom severity. Furthermore, αT supplementation had no effect on serum levels of specific IgE antibodies or lipid peroxides in response to allergen provocation (Montano Velazquez et al. 2006
The prevention of cardiovascular disease (CVD) is one of the major areas in which vitamin E, primarily αT, has drawn a lot of attention in the past decade. While it has long been believed that atherosclerosis and associated CVD is a consequence of oxidative modifications of circulating lipoproteins (LDL in particular), which has been shown to be inhibited by αT in numerous in vitro
studies, it is becoming more evident that atherosclerosis is a consequence of complex interaction between lipid metabolism, vascular function and inflammation (Libby 2002
; Ross 1999
). In a set of studies in both healthy subjects and diabetes patients (which are at higher risk for CVD), αT supplementation was found to have an anti-inflammatory effect on monocytes isolated from supplemented subjects and on in vivo
markers of inflammation. Thus, Devaraj et al.
showed that resting or LPS-stimulated monocytes isolated from normal healthy people supplemented with 1200 IU/d αT for 8 weeks produced less superoxide (O2
), hydrogen peroxide (H2
), lipid peroxides, and IL-1β compared to either baseline or following washout of the supplement (Devaraj et al. 1996
). Stimulated monocytes from αT-supplemented people also displayed a diminished adhesion to vascular endothelial cells in vitro
. In another study, the same investigators tested the effect of αT supplementation on inflammatory parameters in patients suffering from type 2 diabetes. Monocytes isolated from these patients exhibited higher levels of O2
production, IL-1β release, and endothelial cell adhesion upon stimulation with LPS. Supplementation with 1200 IU/d αT for 3 months inhibited the enhanced LPS-induced O2
production, IL-1β release, and endothelial cell adhesion to approximately the same low levels as in normal healthy controls (Devaraj and Jialal 2000a
). αT supplementation also markedly decreased the increased plasma levels of the soluble endothelial cell adhesion molecules VCAM-1, ICAM-1, and E-selectin in diabetic patients. The same authors also reported that αT supplementation decreases plasma levels of C-reactive protein (CRP) in both patients with type 2 diabetes and normal healthy subjects, and that this decrease in CRP is associated with a reduction in LPS-induced IL-6 production in isolated monocytes (Devaraj and Jialal 2000b
). CRP is a hepatic acute phase protein induced by IL-6 and a clinically important marker of ongoing inflammation and predictor of cardiovascular events in humans.
The effect of αT supplementation on plasma CRP and soluble adhesion molecules in type 2 diabetes patients was also studied by other investigators. Thus, supplementation with 800 IU/d of αT for 4 weeks resulted in a two-fold increase in plasma αT and reduced CRP-values by ~50% (Upritchard et al. 2000
). However, soluble ICAM-1 and VCAM-1 concentrations did not change in this study. In another study carried out by Murphy et al.
, a significant reduction of plasma CRP levels compared to placebo was observed in smokers with acute coronary syndrome after 6 months of supplementation with 400 IU/d of αT (Murphy et al. 2004
). CRP levels also decreased in the placebo group, however, and were not any different to those in the αT-supplemented patients for the first few months. Plasma levels of soluble ICAM-1, VCAM-1, E-selectin and P-selectin did not significantly change during the study. In another study, whole blood from either smokers, diabetes type 2 patients, or healthy controls, before and after supplementation with αT (600 IU/d for 4 weeks) was stimulated with LPS and analyzed for the production of TNF-α, IL-1β, and interleukin-1-receptor antagonist (IL-1RA), which counteracts the pro-inflammatory effects of IL-1β. αT supplementation significantly inhibited LPS-stimulated TNF-α and IL-1RA production in whole blood from smokers, but not in whole blood from controls or diabetic patients (Mol et al. 1997
), suggesting that αT may be particularly beneficial to smokers.
Based on the observation that αT supplementation (800 IU/d for ~2 years) reduces composite cardiovascular disease and myocardial infarction in hemodialysis patients (Boaz et al. 2000
), Smith et al.
investigated whether αT supplementation with 400 IU/d for up to 2 months had an effect on plasma markers of inflammation in end-stage renal patients (Smith et al. 2003
). However, αT supplementation had no significant effect on plasma levels of IL-6, CRP, and TNFα, and concentrations of free F2-isoprostanes, a marker of non-enzymatic lipid peroxidation. The only significant effect observed was a nearly 2-fold increase in plasma αT that was associated with a 10-fold increase of its metabolite α-carboxyethyl hydroxychromane (α-CEHC, 2,5,7,8-tetramethyl-2-[2′-carboxyethyl]-6-hydroxychroman) and a significant decrease in γT. In another small clinical trial in dialysis patients with end-stage renal disease, the effect of αT was compared to that of a tocopherol mixture containing 60% RRR
-γT, 28% RRR
-δT and 10% RRR
-αT. Either 300 mg/d of RRR
-αT or the tocopherol-mixture were administered to the patients or healthy subjects for 14 days (Himmelfarb et al. 2003
). Serum concentrations of α- and γT, their metabolites α- and γ-CEHC, IL-6 and CRP were determined as study endpoints. At the onset of the study, αT levels were similar in the two groups, while serum γT levels were significantly higher in hemodialysis patients than in healthy subjects. While αT supplementation neither significantly increased αT in healthy subjects nor in hemodialysis patients (but significantly increased the levels of its degradation product α-CEHC), it lowered γT levels in both groups. In contrast, supplementation with the γT-enriched vitamin E preparation caused a significant increase of γT and its degradation product γ-CEHC in both the normal healthy people and in the hemodialysis patients. While supplement ation with αT had no_effect on CRP concentration in hemodialysis patients, they were significantly lowered by the γT-enriched vitamin E. Interestingly supplementation with αT (but not with the γT-enriched preparation) increased the serum levels of IL-6. In another study, Liu et al
. showed that supplementation of normal healthy people with γT-enriched vitamin E (100 mg γT, 40 mg, δT, and 20 mg αT) inhibited ADP-induced aggregation of isolated platelets more potently than supplementation with pure αT (Liu et al. 2003
). More efficient inhibition of platelet aggregation by the γT-enriched vitamin E was associated with increased constitutive nitric oxide synthase (NOS) activation in platelets. These results suggest that a mixture of tocopherols enriched with γT may be more efficient in inhibiting inflammation-associated disease than αT alone.
2.2. Animal studies
Immune function also declines in animals with age. Similar to the humans studies, LPS-stimulated formation of PGE2
is elevated in macrophages isolated from old mice (24 months of age) compared to young mice (6 months of age) (Wu et al. 1998
). Supplementing old mice with 500 ppm of αT for 4 weeks diminished LPS-induced PGE2
concentrations to levels produced by macrophages from young mice. In contrast, αT supplementation of young mice did not alter LPS-induced PGE2
production compared to unsupplemented controls. αT appears to decrease PGE2
production in macrophages from old mice by inhibiting the elevated cyclooxygenase (COX) activity in these cells (Wu et al. 1998
). The inhibition of the age-related increase of PGE2
may stem from inhibition of lipid peroxidation, which is though to contribute to the enhanced COX-2 expression with age. In a subsequent study, the same authors showed that γT and δT inhibited PGE2
in macrophages isolated from old mice equally well or even more potently than αT, whereas βT had no effect (Wu et al. 2000
). In contrast, splenocyte IL-2 production was not affected by either αT or βT, but was increased by γT and δT. These results indicate that the different forms of tocopherol have different effects on immune function.
To see whether the immunostimulatory effect of αT has any impact on the course of a viral infection, both 4 and 22 month-old C57BL/6NIA mice were infected with influenza A/Port Chalmers/1/73 (H3
) virus after feeding them for 6 weeks with a diet containing either 500 ppm or 30 ppm αT acetate. Viral titers were determined at 2, 5, and 7 days post-infection. In young mice, supplementation with high αT levels had no significant effect on pulmonary virus titers compared to the animals fed the low αT diet (i.e.
, controls), except for a slight reduction on day 5. However, in old mice, pulmonary viral titers were up to 25-fold lower in animals fed with the high αT diet during the course of infection, with titers being in the same range as in young animals (Hayek et al. 1997
). Subsequently, Han et al.
investigated whether the inhibitory effect of αT on viral titers in old mice was due to a change in the production of the Th1 cytokines IFN-γ, IL-2, IL-6, and TNF-α (Han et al. 2000
). Indeed, αT supplementation enhanced IFN-γ levels in old mice on day 5 and 7 post infection to the same high levels as in young mice. Overall, higher IFN-γ levels correlated with lower pulmonary viral titers. IL-2 concentrations were partially restored by αT supplementation. In contrast, αT administration had no effect on IL-2 and IFN-γ in young mice. There was no significant overall effect of age, αT supplementation, or infection on splenocyte IL-4 or IL-6 production. Macrophages isolated from old mice produced significantly higher levels of PGE2
in response to LPS, which was diminished by supplementation with αT to levels produced by macrophages from young mice. These results strongly suggest that αT decrease pulmonary influenza virus titers in old mice by inhibiting the elevated production of PGE2
, and by at least partially restoring the attenuated Th1 cytokine response in these animals.
IgE antibodies are involved in mediating the type 1 hypersensitivity response in allergic reactions. In a murine nasal allergy model, Zheng et al.
showed that supplementation with a diet containing 535 mg αT per kg for 4 weeks significantly reduced quantifiable nasal symptoms compared with a diet containing normal levels of αT (Zheng et al. 1999
). In contrast to the human studies discussed previously, αT supplementation also led to a significant decrease in lymphocyte proliferation, serum IgE levels and production of the Th2 cytokines IL-4 and IL-5 in these animals. In another study, Bando et al.
tested the effect of vitamin E treatment in an allergic mouse model (Bando et al. 2003
). Ovalbumin-specific IgE antibodies were analyzed by passive cutaneous anaphylaxis reaction. Different all rac
-αT concentrations (0.5, 5, 10 and 50 mg/100 g diet) were used to supplement 6 week old BALB/c mice for a total of 6 weeks. 21 and 35 days after the onset of supplementation, mice were immunized with ovalbumin and bled 1 week after the second immunization. The isolated sera were subcutaneously injected at different concentrations into rats, and 2 days post sensitization, ovalbumin and Evans blue injected and the blue reaction spots evaluated. Sera from control-fed animals (i.e.
, 5 mg/100 g diet) elicited the strongest immune response, while those from animals fed with 10 and 50 mg αT/100 g diet, showed positive spots only at the highest dilution and were smaller in appearance. A decrease in reactive IgE antibodies was also observed at 0.5 mg/100 g diet, which was ascribed to a generally decreased immune response due to vitamin E deficiency.
Jiang and Ames have tested the relative anti-inflammatory efficacy of γT and αT in the air-pouch inflammation model, which is thought to mimic human joint disease. Inflammation is induced by a single injection of the long-chain sulfated polysaccharide carrageenan into the intrascapular area of male Wistar rats. γT (33–100 mg/kg body weight) or γ-CEHC (2 mg in pouch), but not αT (33 mg/kg), significantly inhibited carrageenan-induced PGE2
accumulation at the site of inflammation (Jiang and Ames 2003
). γT administration also significantly inhibited formation of LTB4
, a potent leukocyte chemoattractant, suggesting that γT may inhibit 5-lipoxygenase activity. In addition to LTB4
and other pro-inflammatory eicosanoids, γT supplementation also led to a reduction of TNF-α at the site of inflammation. γT administration also attenuated inflammation-mediated oxidative damage as indicated by a significant reduction of 8-isoprostane (an F2 isoprostane), and lactate dehydrogenase, as a marker of tissue damage (Jiang and Ames 2003
). γT also significantly attenuated the partial loss of food consumption caused by the strong inflammatory reaction (Jiang and Ames 2003
In a study in rats, in which peritonitis was induced by i.p.
injection of zymosan, we found that moderate γT supplementation (90 mg/kg diet) attenuated inflammation-induced protein nitration and ascorbate oxidation, as indicated by a significant reduction of protein-bound 3-nitrotyrosine and dehydroascorbate formation in the kidney, compared to animals that received a normal αT-containing diet (Jiang et al. 2002
). Supplementation significantly attenuated inflammation-induced loss of vitamin C in the plasma and kidney. Interestingly, in untreated control animals, γT supplementation lowered basal levels of 3-nitrotyrosine in the kidney and buffered the starvation-induced changes in vitamin C in the examined tissues. Similarly, Takahashi et al.
recently showed that γT, but not αT, potently inhibited neointimal formation induced by vascular injury in the insulin-resistant rats (Takahashi et al. 2006
). Along with vascular protection, γT, but not αT markedly reduced the increased presence of 3-nitrotyrosine in neointimal tissue in insulin-resistant rats. In another study, Milatovic et al
. found that i.p.
administration of γT (100 mg/kg body weight) significantly inhibited LPS-induced cerebral PGE2
generation in rats (Milatovic et al. 2003
). However, supplementation did not significantly attenuate LPS-induced neuronal oxidative damage or dendritic degeneration in the brain. The relatively moderate inhibition of PGE2
and lack of protection may be due to the relatively low bioavailability of γT in the brain after i.p.
administration, which was not assessed, however.
As with the human studies, Saldeen and his coworkers found that supplementation of Sprague Dawley rats with γT-enriched tocopherol led to a more potent decrease in platelet aggregation and delay of arterial therombogenesis compared to supplementation with αT (Saldeen et al. 1999
). Supplementation with the γT-enriched tocopherol also resulted in stronger ex vivo
inhibition of superoxide generation, lipid peroxidation and LDL oxidation. In a follow-up study, they reported that γT-enriched tocopherol was significantly more potent than αT in enhancing SOD activity in plasma and arterial tissue, as well as increasing arterial protein expression of both MnSOD and Cu/Zn SOD (Li et al. 1999
). Furthermore, although both tocopherols increased NO generation and endothelial nitric oxide synthase (eNOS) activity, only supplementation with the tocopherol mixture resulted in increased protein expression of eNOS.
Finally, Yoshida et al.
recently demonstrated that topical application of 5% γ-tocopherol-N, N
-dimethylglycinate hydrochloride, a hydrophilic γT derivative, either before or after UV irradiation significantly attenuated PGE2
synthesis and edema formation (Yoshida et al. 2006
). Although the pre-application of 10% αT acetate had a similar anti-inflammatory effect as that of the γT derivative, post-treatment of αT was ineffective in comparison. The authors also showed that administration of the γT derivative more potently inhibited COX-2 activity than αT, while αT caused a more extensive downregulation of COX-2 protein in pre-treated animals.
In summary, the animal studies show that both α- and γT have significant anti-inflammatory activity, supporting the observations made in the humans studies, but that their relative potencies vary depending on the system tested. An explanation for these differences may be they impact on different molecular targets.