Influence of Vitamins E and B, on Immune Response SIMIN NIKBIN MEYDANI, MICHAEL HAYEK, AND LAURA COLEMAN Nutritional Immunology Laboratory USDA Human Nutrition Research Center on Aging at Tufrs University 71I Washington Street Boston, Massachusetts 02111

INTRODUCTION Several micro- and macronutrients are needed for normal maintenance of the immune system. These include amino acids, essential fatty acids, and several vitamins and minerals. Numerous investigations since the early 1900s have shown that marginal and profound nutrient deficiencies impair the immune response in animal models as well as in humans so that the dependency of the immune system on adequate nutritional status is unquestionable. More recent studies, however, have looked beyond deficiency of nutrients and focused on the level of nutrients needed for optimal immune response. Evidence indicates that for specific nutrients and among certain subgroups of the population, levels higher than the recommended dietary allowance (RDA) are needed to maintain optimal immune response. Vitamins E and B, are two such nutrients. Several reviews have focused on the effects of vitamin B6 and E deficiencies on the immune response,'-4 and thus they will not be extensively reviewed here. Rather, this paper will focus on studies that have investigated the role of supplementation with levels greater than the RDA for these nutrients. VITAMIN E AND IMMUNE RESPONSE Vitamin E Deficiency

Several studies have shown that vitamin E deficiency impairs both cellular and humoral immunity in different animal models (reviewed in refs. 2-4). This impairment of the immune response is associated with increased incidence of disease^.^.^,^ Similarly, we found that vitamin E deficiency in humans impairs T cell-mediated function, a defect that is reversible by vitamin E supplementation.' Vitamin E Supplementation: Animal Studies

Vitamin E is one of the few nutrients for which supplementation using higher than recommended levels has been shown to enhance the immune response. The beneficial effects of supplemental vitamin E above the recommended dietary levels 125

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have been recognized since the 1950s when Segani' observed that rabbits supplemented with vitamin E produced antibodies earlier to typhoid vaccine, staphylococcus, and 0-streptolysin than those fed control diets, although no difference in peak antibody level was found between the two groups. Later, Tengerdy et al.' reported that vitamin E supplementation (150-300 mg/kg) of chickens fed a commercial stock diet (43 mg/kg of vitamin E) increased survival against Escherichiu coli ( E . coli) infection. The increased protection correlated with elevated hemagglutination (HA) titer and faster clearance of E . coli from the blood. Since these initial reports, a host of studies, in a variety of species, have demonstrated the immunoenhancing potential of supplemental vitamin E. In mice it has been demonstrated that in uitro addition of vitamin E to lymphocytes can act directly as a mitogen, as well as enhance lymphocyte proliferation by other mitogens such as lipopolysaccharide (LPS), concanavalin A (Con A), and phytohemagglutinin (PHA).".'" Bendich et al." demonstrated that although a 15 mg/kg diet of vitamin E was adequate to prevent myopathy in spontaneously hypertensive rats, optimal lymphocyte proliferation to PHA and Con A was only obtained with much higher levels of vitamin E (50-200 mg/kg). Tanaka et ul.l'demonstrated that dietary vitamin E was able to enhance helper T-cell activity, whereas Moriguchi et u1.I' showed that supplementation of rats with 500-2500 ppm of vitamin E for 10 days increased mitogenic response of splenocytes to Con A and LPS, increased natural killer (NK) cytotoxicity against YAC- 1 cells, and increased phagocytic activity of alveolar macrophages in a dose-dependent manner. These, as well as studies reported in many other species, demonstrate that dietary vitamin E above currently recommended levels can enhance various aspects of the immune response.

Vitamin E Supplementation: Human Studies

In humans, evidence is also accumulating for an interactive effect of supplemental vitamin E and the immune system (TABLE1). Several studies have demonstrated that vitamin E can affect polymorphonuclear cell (PMN) function. Baehner et al.I4 evaluated PMN function in three volunteers before and after receiving 400 IU of vitamin E for several days. Although vitamin E was shown to enhance phagocytic rates, it was also shown to decrease bactericidal activity. The authors suggested that the enhanced phagocytic uptake of particles by vitamin E-enriched PMN was associated with the release of increased quantities of 0; anion but markedly reduced quantities of H202.which correlated with diminished bactericidal killing. A depression of bactericidal activity was also reported in a study by Prasad15 in which he examined 13 adult males (24-30 y) and 5 young boys (13-18 y) receiving 300 mg dl-a-tocopheryl acetate/d for three weeks. Interestingly, two subjects showed clinical improvement in their symptoms of asthmatic attacks and nasal allergy. Richards et 0 1 . ' ~showed that supplementation of smokers with 900 1U of vitamin E for six weeks caused a transient decrease in chemiluminescence. They attributed this to vitamin E's ability to inhibit the generation of oxidants by activated phagocytes. In order to study the depressive effect of pharmacological doses of vitamin E on PMN oxidative metabolism, Engle et a1.I7 examined 0; anion production by PMN following in uitro exposure to pharmacological doses of vitamin E. Superoxide anion production of PMN after a 5 min exposure to phorbol myristate acetate

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u7

(PMA) was significantly decreased in the vitamin E-treated PMN compared to vehicle control PMN. This decrease in superoxide anion production, however, was associated with 5.0 and 10.0 mg/dL (pharmacological doses; difficult to obtain with in vivo supplementation) but not with 3.5 mg/dL (high levels of vitamin TABLE 1. Effect of in vivo Vitamin E Supplementation on Human Immune

Response Subject and Dosage healthy young 400 IU/d < I wk healthy young 300 IU/d for 3 wk chronic care facility; elderly 200 or 400 IU/d for 1 year institutionalized healthy elderly 200 IU/d for I year healthy elderly 800 IUfd for 30 d

healthy elderly 800 IU/d for 60 d healthy young and elderly 800 IU/d for 60 d

Immune parameter PMN PMN Mitogenic response DTH Ab development to influenza virus vaccine

IgG and C3 levels

Mitogenic response IL-I IL-2 DTH Neutrophils IL- 1 TNF IL-6

Effect Phagocytic rate

t

J Bactericidal activity

1

Literature cited Baehner et Prasad”

et H

Harmon & Miller31

T

Ziemlanski et a/.”’.‘‘

Meydani et a/.” c,

t t Prevented neutrophilia during exercise

Cannon et

Prevented exercise-induced rise

Cannon et a/.34

4-3

1 t

young smokers 900 IU/d for 6 wk

DTH PMN

healthy young 200 IU/d for 8 wk

PBMC

Prevented fish-oil-induced suppression of Con A mitogenesis

Kramer et al.”

long-stay elderly 50 IU/d for 28 d

Mitogenic response toFHA .

t

Penn et

a

Richards et

chemiluminescence activity

et

In conjunction with vitamin C. In conjunction with vitamins A and C.

E ; obtainable with in vivo supplementation). In another study, Okano et supplemented 19 college students with 600 mg a-tocopherol daily for three months. They found no difference in the chemiluminescence of the PMN from the supplemented subjects compared to controls. They reported that the vitamin E concentra-

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12x

tion in the PMN of the supplemented subjects never exceeded 3.5 pg/109 cells. These studies suggest that the concentration, as well as the duration, of vitamin E supplementation may account for the effect that vitamin E exerts on PMN. Several other studies have demonstrated that in uitro addition of vitamin E can also affect the function of different cells of the immune system (TABLE2 ) . Villa et reported that vitamin E supplementation in vitro caused a concentrationdependent inhibition of arachidonic acid-induced aggregation of PMN and mononuclear leukocytes (MNL). They suggested that this may have been due to interference with lipoxygenase activity. Topika et a/.” found that the addition of a tocopherol (0.2 mM/L) to human lymphocytes in uitro suppressed lipid peroxidation and oxidant damage to DNA when induced by the catalytic system of Fez+sodium ascorbate. Sepe and Clarke” studied neutrophil lysis in liposome target vesicles. They demonstrated that stimulated neutrophils secrete myeloperoxidase and H 2 0 Z ,which combine with extracellular halides to form a membrane lytic system. In uifroaddition of a-tocopherol to the liposomes increased their resistance to myeloperoxidase-generated H20, exposure. Leb et a/.?‘ reported that in uitro addition of a-tocopherol to monocytes inhibited PMA-induced cytotoxicity. This inhibition was due to an a-tocopherol-related decrease in monocyte release of H,02. a-Tocopherol, however, was not effective in reducing monocyte antibody-dependent cytotoxicity (ADCC), presumably because nonoxidative injury is more important in ADCC.

Effect of in Vitro Vitamin E Supplementation on Polymorphonuclear Cell Function in Humans

TABLE. 2.

Immune parameter Neutrophils Neutrophils

Effect

Literature cited

resistance to H20! PMA-induced cytotoxicity ADCC 1-0; production dependent on vitamin E level chemiluminescence

Leb e I a/.” Sepe and Clark?’

t

++

PM N PM N

++

Engle et

01.‘’

Okano e l a/.’*

Vitamin E Supplementation: Aging Immune Response

Considerable evidence indicates that aging is associated with altered regulation of the immune system.?’ The most dramatic effect is on cell-mediated immunity,?4-?6One of the biological changes associated with aging is increased free radical formation with subsequent damage to cellular processes. Numerous studies have focused on the free radical theory of aging and the role of antioxidants, including vitamin E, on the life expectancy of rodents. Oxygen metabolites, especially H,02, produced by activated macrophages, depress lymphocyte proliferation.27Free radical formation associated with aging may be an underlying factor in the depressed immune response. Therefore, tocopherol supplementation during aging might result in the enhancement of the immune response. Several studies have suggested that vitamin E supplementation will enhance immunity in the aged (TABLE1). We have previously shown that splenocytes from

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old mice fed 500 ppm dl-a-tocopheryl acetate for six weeks had significantly higher proliferative response to Con A and LPS than old animals fed 30 ppm vitamin E.** In addition, vitamin E supplementation significantly increased delayed-type This immunostimhypersensitivity (DTH) skin test to 2,4-dinitro-7-fluorobenzene. ulatory effect of vitamin E was associated with increased production of interleukin (IL)-2 and decreased production of prostaglandin (PG) E,. Harmon et also demonstrated improved immune response and survival in vitamin E-supplemented, short-lived autoimmune prone NZB/NZW mice compared to control mice. supplemented institutionalized healthy elderly women with Ziemlanski et vitamin E and vitamin C (400 mg/day) and assessed serum proteins and immunoglobulin concentrations after 4 and 12 months. Vitamin E increased total serum protein, with the principal effect on a-2- and p-2-globulin fractions occurring at 4 months. No significant effects were noted in the levels of either immunoglobulins or complement C3 after 12 months, although there was a significant increase in serum protein concentration. The group that had been administered vitamin C and vitamin E, however, displayed significant increases in IgG and complement C3 levels. In another study, Harmon and Mille$' supplemented 103 elderly patients from a chronic care facility with 200 or 400 IU/d a-tocopheryl acetate for 12 months. No beneficial effect on antibody development against influenza virus vaccine was observed. Unfortunately, data on health status, medication use, antibody levels, and other relevant parameters were not reported. Meydani et al. ,32 using a double-blind placebo controlled study, supplemented 32 healthy men and women ( 2 60 years of age) with either a placebo or 800 mg DLa-tocopheryl acetate (400mg capsules 2 times daily) for 30 days. DTH, mitogenic response, IL-1, IL-2, and PGE, formation were evaluated. Vitamin E-supplemented subjects showed increases in plasma vitamin E level, DTH score, mitogenic response to Con A, as well as IL-2 production (FIG. 1, TABLE3). Vitamin E supplementation was also associated with decreases in PHA-stimulated PGE, production by peripheral blood mononuclear cells (PBMC) as well as plasma lipid peroxide levels (TABLE3). Vitamin E supplementation had no effect on mitogenic response of PBMC to PHA or on IL-1 production. Exercise has been shown to increase oxidative stress, as well as production of inflammatory cytokines, such as JL-1. We therefore studied the interaction between vitamin E supplementation and exercise on cytokine production by young and old v o l ~ n t e e r s Young . ~ ~ (22-29 yr) and old (55-74 yr) subjects were supplemented with 800 IU of dl-a-tocopheryl acetate for 60 days before undergoing eccentric exercise (downhill running). Although vitamin E did not affect the preexercise production of IL-1 or tumor necrosis factor (TNF), it prevented the eccentric exercise-induced rise of IL-1. Vitamin E supplementation did, however, inhibit the production of another cytokine, IL-6. Furthermore, young subjects fed placebo capsules had significantly greater neutrophilia and plasma creatine kinase levels in response to eccentric exercise, compared to old subjects receiving placebo. Vitamin E supplementation eliminated the difference in neutrophilia and creatine kinase level between the two age groups.34Because IL-1 and IL-6 have been implicated in the inflammatory process and acute phase response, and IL- 1 has been indicated in exercise-induced muscle proteolysis and damage, their inhibition by vitamin E during damaging exercise could have practical implications. These studies indicate that in addition to its enhancement of cell-mediated immunity, vitamin E, by its modulation of cytokine production, can affect the catabolic consequences of the inflammatory process and acute phase response. ~ that elderly long-stay patients who were supplemented Penn et ~ 1 . 'reported

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130 Jmmd

*

A

T xmo-

lmm

-

0-r

placebo

vitmin E

placebo

vitamin E

*

C

T

vibmin E

placebo

Treatment

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of Vitamin E Supplementation on ex Viuo PGE, Production by PBMC, Plasma Tocopherol, and Lipid Peroxide Levels in Older Adults"

TABLE 3. Effect

Placebo Plasma a-tocopherol (pmol/L)

before after

PBMC PGE,b (In pmol/L) Plasma lipid peroxide

26.2 23.9

Vitamin E

2 1.7 ? 1.4

23.9 t 1.4 70.9 2 6.3'

before after

8.3 2 1.6 8.4 ? 1.6

9.1 1.6 8.5 ? 1.5'

before after

2.3 2 0.8 2.2 ? 0.6

2.8 2 0.7 1.2 f 0.6'

*

" Mean 5 SEM; healthy elderly supplemented their diet with 800 IU/d dl-a-tocophoryl acetate for 30 days in a double-blind, placebo-controlled, residential trial (adapted from Meydani et a/."). * 1 x 10' to 5 x 10' cells/mL were stimulated with 2.5 to 10 mg/mL PHA for 48 h. PGEz in supernatant was measured by radioimmunoassay. Significantly different from before values by paired Student's t test or paired Wilcoxon's signed-rank rest. with a mixture of vitamin E (50 mg), vitamin A (8000 IU), and vitamin C (100 mg) for 28 days showed improvement in the percentage of helper T cells (CD4+) and the mitogenic response to PHA, compared to those receiving the placebo. The improvement observed in this study, however, cannot be solely attributed to vitamin E, as vitamin C and vitamin A also are needed for normal immune response. Furthermore, 5% of subjects were vitamin E-deficient; but 21% were deficient in vitamin A, and 47% were deficient in vitamin C. The deficiencies of vitamins E and A were corrected, although that of vitamin C was not corrected.

Vitamin E Supplementation: Environmental Oxidants and Immune Response Levels of tocopherol above the RDA might also be needed for optimal immune response when oxidative stress is increased due to environmental factors such as high oxidative exposure, oxidizing pollutants, or consumption of pro-oxidant '~ that a combination of vitamin E deficiency nutrients. Eskew et ~ 1 . demonstrated and ozone exposure in rats resulted in depression of ADCC, whereas the same treatments administered singly had no effect. Fish oil supplementation, rich in (n-3) polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) has been shown to decrease various aspects of the immune system, particu-

FIGURE 1. Effect of vitamin E supplementation on immune response of older adults. Data is presented as mean ? SEM. Healthy elderly supplemented their diet with 800 IU/d of dla-tocopheryl acetate for 30 days in a double-blind, placebo-controlled residential trial (adapted from Meydani et a/."). A: 1 x lo6 cells/mL were cultured in presence or absence of optimal concentration of Con A for 68 h. Cell proliferation was measured by incorporation of [3H]thymidine into newly synthesized DNA following a 4 h pulse. Values represent corrected counts per minute, which is the cpm of stimulated culture minus the cpm of unstimulated cultures. B: 1 x lo6 cells/mL were cultured in the presence of Con A for 48 h. IL-2 production was measured in supernatant using a bioassay. C: DTH was measured using Multi-Test CMI; data represent cumulative index, which is the sum of induration diameter of all positive responses. Asterisk indicates significantly different from before values.

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larly in the e l d e r l ~ .It~is~postulated .~~ that the fish oil-induced immunosuppression is due to an increased tocopherol requirement.j9 This is supported by the recent study of Kramer et al,40who investigated whether increased vitamin E intake would restore the fish oil-induced suppression of T-lymphocyte blastogenesis. In their study, healthy men were supplemented for a ten-week period with fish oil (15 mg/d; 50% concentrate of refined anchovy oil containing 50% (n-3) fatty acids) followed by an eight-week period of fish oil plus 200 mg a-tocopherol/day. Fish oil supplementation depressed the PBMC response to Con A, an effect that was reversible by vitamin E supplementation. A positive correlation between a-tocopherol concentrations and responsiveness of T lymphocytes to Con A was also demon strated .40

EPIDEMIOLOGICAL STUDIES Epidemiological studies have yielded mixed conclusions about the interaction between vitamin E and immunity. Goodwin and Garry4I found no positive correlation between vitamin E intake and DTH, mitogen stimulation, serum antibodies, or circulating antibodies in healthy adults (65-94 years) consuming megadoses of . ~ a ~negative correlation between dietary different vitamins. Payette et ~ 1reported vitamin E and ex uivo IL-2 production. These studies, however, suffer from complications of using several vitamin supplements at megadose levels, which can confound the findings due to possible nutrient interaction^,^' inaccurate measures . ~ ~the other hand, of vitamin E status, or an ill-defined subject p ~ p u l a t i o n On ~ ~ . a ~positive ~ correlation between plasma vitamin E level Chavance L't ~ 1 . reported and DTH response to diphtheria toxoid, candida, and trichophyton in 100 healthy elderly subjects over 60 years of age. Subjects with tocopherol levels greater than 135 mglL were found to have higher helper-inducedicytotoxic-suppressorratios. Blood vitamin E concentrations were also negatively correlated with the number of infectious disease episodes in the preceding year.

VITAMIN B, AND THE IMMUNE RESPONSE Vitamin B, Deficiency Vitamin B, deficiency has been shown to impair both humoral and cell-mediated immunity in rodents. Vitamin B,-deficient mice, rats, and guinea pigs showed impaired primary and secondary antibody production and reduced numbers of antibody-producing cells, as well as DTH and graft versus host r e a ~ t i o n . ~A~ - ~ ~ dramatic depletion of thoracic-duct lymphocytes, and reduced in vitro lymphocyte pr~liferation~~~"' and T cell-mediated c y t o t o ~ i c i t y ~were ~ . ~ 'also reported in vitamin B,-deficient animals. Very few studies, however, have addressed the effects of vitamin Bh deficiency on immune response of humans. Although isolated vitamin Bh deficiency is rare in humans, particular subgroups of the population, such as the elderly, may be at increased risk for developing deficiency of vitamin B6.S2,53 Cheslock and M ~ C a l l y ' reported ~ a decrease in the number of circulating lymphocytes in humans consuming low vitamin B, diets or treated with deoxypyriAntibody production was also reduced in the doxine (a vitamin B, antagoni~t).~' ~ a modest but significant study by Vilter et al.55Van den Berg et 0 1 . ~reported decreae in the percent helper T cells and serum immunoglobulin D levels following

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133

marginal vitamin B, deficiency. The impairment of the immune response in alcoholi c ~ and ~ ' in patients with chronic renal failure has also been attributed to vitamin B6 d e f i c i e n ~ y . ~ We6' * . ~ ~ recently reported a significant decrease in lymphocyte number, IL-2 production, and lymphoproliferative response to mitogens in healthy elderly adults following 21 days of consuming a vitamin B,-depleted diet (TABLE 4 and FIG.2).

Vitamin B, Supplementation

There have been few studies on the effects of vitamin B, supplementation on the immune system in humans. Goodwin and Garry4' studied the immunological effects of meganutrient supplementation (intakes greater than five times the RDA) in 270 healthy elderly (65-94 years old) subjects. Forty-six percent of these individuals were taking vitamin B, supplements, either alone, or in combination with

Effect of Vitamin B, Status on White Blood Cell (WBC) and Lymphocyte Number, and on ex Viuo IL-2 Production of Healthy Older Adults ' I

TABLE 4.

WBC Time

BL~ - B6 + B& + BJI + BnIII Final

10'/mm3

6.45 6.01 6.13 6.14 6.48 6.51

2 0.56

0.44 t 0.61 2 0.51 2 0.67 ? 0.73 -C

Lymphocyte Percent

Lymphocyte I~~/mrn'

IL-2 kU/L

42.00 t 4.30 34.38 2.80' 34.50 4.40 37.38 ? 2.49 34.63 '. 2.05 36.14 3.14'

2.69 2 0.28 2.08 t 0.24' 1.97 0.28 2.29 ? 0.21 2.21 0.23 2.30 0.35

104 17 41 13 55 t 31 43 9 93 57 142 2 70

* * *

*

*

* * * *

Mean -+ SEM; n = 8. Values represent baseline (BL), vitamin B,-deficiency stage (-861, the three stages of vitamin B, repletion ( fBJ, + B611, and + BJU, and the final stage of the study. Data adapted from Meydani el trl." 1 Significantly different from baseline, p < 0.05. "

other vitamins and minerals.6' The range of vitamin B, intake for subjects in the upper quartile of total intake, for example, was from 2.5 to 182 times the RDA. These subjects, compared to those in the bottom 75% had significant decreases in mean lymphocyte counts. There were no significant differences, however, between individuals with high and low vitamin B, intakes with respect to mean PMN counts, DTH, serum autoantibodies, or lymphocyte proliferation in response to PHA stimulation. The authors concluded that the immunological benefits that others have observed as a result of nutrient supplementationb2may, in fact, be only a short-term effect that diminishes or disappears with time. Neither the length of time, however, that individuals in this study were taking vitamin supplements, nor the consistency with which they took supplements was clearly stated, so interpretation of these results is difficult. Additional studies of vitamin B, supplementation have been done in uremic patients, in whom immunological abnormalities are often observed. Dobbelstein eta/.58found that supplementation with 300 mg per day of pyridoxine hydrochloride

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(PN HCl) for two weeks in patients with chronic uremia restored activation coefficients (as a measure of vitamin B, status) to normal and significantly increased the reactivity of these patient's lymphocytes in mixed lymphocyte cultures. The authors hypothesized that uremic toxins may exert an inhibitory effect on pyridoxal kinase, the intracellular enzyme needed for the phosphorylation of pyridoxal and its analogues. Pyridoxal-5' phosphate (PLP) is necessary for normal cellular immune responses, and depletion of this compound could lead to subsequent immunological impairment. Casciato et a/.63administered 50 mg of PN HCl per day for three to five weeks to eight patients on maintenance hemodialysis and observed an increase in the

T

BL

-B6

+B6I

+B6II

+B611I

Final

Study Phase FIGURE 2. Effect of vitamin B, status on mitogenic response of PBMC from healthy older adults to optimal dose of Con A. Data are expressed as percent response of control subjects tested on the same day as experimental subjects; mean 2 SEM, n = 8. Data adapted from Meydani et a/.69Asterisk indicates significantly different from baseline at p < .05.

generation of chemotactic factors from plasma as well as an increase in in uitro lymphocyte proliferation in response to mitogenic stimulation. Kleiner et al. ,M however, found no effect of 300 mg/day PN HCl supplementation among hemodialysis patients with respect to lymphoproliferative response to the mitogens PHA or Con A. This may, however, have been at least partially related to the fact that lymphocyte proliferation responses and vitamin B6 status of these patients were both normal before PN supplementation. Results regarding the impact of vitamin B, supplementation on immune function in patients with chronic renal failure seem inconclusive to data. In healthy elderly subjects, Talbott et a1."* studied the effect of pyridoxine

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supplementation on lymphocyte responsiveness in 15 free-living women. Subjects received either 50 mg/day of PN HCl (equivalent to 41.13 mg of PN) or a placebo for two months. Lymphocyte proliferation to both T- and B-cell mitogens, lymphocyte subpopulations with monoclonal antibodies, and plasma PLP were measured at baseline after one month and after two months of either PN or placebo supplementation. Plasma PLP levels increased, and lymphocyte proliferation increased significantly in response to PHA, pokeweed mitogen (PWM), and Staphylococcus aureus (SAC). For PN-treated subjects with low baseline plasma PLP levels, lymphocyte proliferation also increased significantly in response to Con A. Percentages of T3+ and T4+ but not T8+ cells increased significantly in subjects treated with PN supplements. The researchers suggested that improving vitamin B, status in the elderly is important in stimulating immunocompetence. The amount of PN that the subjects received (50 mg PN HCl/day), however, was several orders of magnitude greater than the RDA, or a physiological intake. Although acute toxicity of vitamin B, is low, when taken in gram amounts for several months, ataxia and a severe sensory neuropathy can occur.65 Although the amount of PN provided in this study may not have been enough to cause toxicity symptoms, this level of intake is still too high to be recommended on a long-term basis. Therefore, despite the statistically significant results found from vitamin B, supplementation in this study, the practical significance of such findings must be questioned. Most recently, Meydani et a/.60investigated t h e effect of vitamin B, depletion and repletion on immune response in eight healthy elderly adults. This was done as part of a larger study to establish the vitamin B, requirement of older subjects.66 The protocol consisted of a five-day baseline period, a vitamin B, depletion period of 5 2 0 days, three 21-day stages of vitamin B, repletion, and a four-day final phase. The amounts of vitamin B, (given as PN HCl) ingested during the depletion and repletion phases of the study were 3.00, 15.00,22.50, and 33.75 pg/kg body weight per day (0.17, 1.34, 1.96, and 2.88 mg/day for men, respectively, and 0.10, 0.89, 1.29, and 1.90 mg/day for women, respectively). During the final phase, subjects received 50 mg vitamin B6 per day. Vitamin B, status during each stage of the study was assessed by measuring the 24-hour urinary excretion of xanthurenic acid after a 5-gram L-tryptophan load, red-blood-cell aspartate aminotransferase activity coefficients, 24-hour urinary, 4-pyridoxic acid, and plasma PLP. Vitamin B, depletion significantly decreased the percentage and total number of lymphocytes, mitogenic responses of peripheral blood lymphocytes to T- and B-cell mitogens (PHA, Con A, and SAC), and IL-2 production. For most subjects, but not all, these parameters returned to baseline levels after the third repletion phase, when the total vitamin €3, intakes were 1.90 mg/day for women and 2.88 mg/day for men. The percentage and total number of lymphocytes, however, did not return to baseline levels, even after the final supplementation period (see FIG. 2 and TABLE 4). These findings suggest that for maintenance of normal immune function, older adults may require amounts of vitamin €3, higher than those currently recommended. It is not certain, however, whether this higher requirement is specific to the elderly, or whether it is a more generalized effect, regardless of age.60 This study is important because physiological levels of vitamin B, were used, except during the final four-day supplementation period. The short duration of supplementation after repletion, however, may have prevented seeing additional benefits that others6*have reported. The mechanism of immunosuppression induced by vitamin B, depletion was not addressed in this study. The observed decreases in 1L-2 production and lymphocyte proliferation may have been partially due to a significant decrease in both the percentage and absolute number of lymphocytes (particularly T-helper cells,

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which produce IL-2) during vitamin B6 depletion. A decrease in the T-helper cell population could explain the decreased IL-2 production and subsequent decreased mitogenic responses. Furthermore, both of these normal immune responses depend on DNA, RNA, and protein synthesis; vitamin B6 acts as an important cofactor in the production of one-carbon units from serine during nucleic acid synthesis. Impaired nucleic acid synthesis during vitamin B, deficiency could lead to decreased IL-2 production and consequently decreased lymphocyte proliferation.

CONCLUSION Vitamins E and B6 are required for the normal maintenance of the immune response. Deficiencies of both nutrients impair the immune response and decrease resistance to diseases. Recent evidence indicates that optimal function of the immune system might require higher than RDA levels of these nutrients, at least in certain age groups of the population. The elderly are at greater risk for low consumption of these nutrients and exhibit lower immune responsiveness compared to young subjects. Recent studies indicate that supplementation with higher than RDA levels of vitamins B6 and E improves the immune response in older adults. Further research is needed to determine the appropriate levels of vitamins E and B6 needed to maintain optimal immune responsiveness in older adults. Animal and epidemiological studies indicate that this improvement in immune response is associated with increased resistance to diseases and thus can have practical implications in improving general health status and quality of life for older adults. REFERENCES I. 2.

3. 4.

5. 6. 7. 8. 9. lo.

AXLEROD,A. E. 1971. Immune processes in vitamin deficiency states. Am. J. Clin. Nutr. R. P., R. H. HEINZERLING & M. M. MATHIAS.1978. Effect of vitamin E TENGERDY, on disease resistance and immune responses. In Tocopherol, Oxygen, and Biomembranes. C. de Duve & 0. Hayaishi, Eds.: 191-200. ElsevierlNorth-Holland Biomedical Press. Amsterdam. BENDICH,A. 1988. Antioxidant vitamins and immune responses. In Nutrition and Immunology. R. K. Chandra Ed.: 125-148. Alan R. Liss Inc. New York. S. N. & J . B. BLUMBERG. 1991. Vitamin E supplementation and enhanceMEYDANI, ment of immune responsiveness in the aged. In Micronutrients in Health and in Disease Prevention. Adrianne Bendich & C. E. Butterworth, Jr. Eds.: 13: 289-306. Marcel Dekker. New York. JULSETH, D. R. 1984. Evaluation of vitamin E and disease stress on turkey performance. M. S. Thesis. Colorado State University. Fort Collins, CO. G . L., L. S. JENSEN& P. L. LONG.1984. Effect of selenium and vitamin E COLNAGO, on the development of immunity to coccidiosis in chickens. Poult. Sci. 63:1136-1 143. KOWDLEY, K. V., S. N. MEYDANI, S. CORNWALL, R. J . GRAND& J. B. MASON.1992. Reversal of depressed T-lymphocyte function with repletion of vitamin E deficiency. Gastroenterology. 102: 2 139-2 142. E. 1955. Vitamin E effect on vaccination. Minerva Pediatr 7: 985-987. SEGAGNI, CORWIN,L. M. & J . SHLOSS. 1980. Influence of vitamin E on the mitogenic response of murine lymphoid cells. J. Nutr. 110 916-923. CORWIN,L. M. & J. SHLOSS.1980. Role of antioxidants on the stimulation of the mitogenic response. J. Nutr. 110 2397-2422.

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DISCUSSION UNIDENTIFIED SPEAKER: Did you look at the beta-carotene levels in your patients that were fed vitamin E? Beta-carotene has some effect on immune function, and you might get preservation with the vitamin E in the plasma.

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S. N. MEYDANI(Tuffs University, Boston, M A ) : Yes; but we didn’t see any effect. We look at not only beta-carotene but vitamin A, vitamin C, and several other nutrients. None of the nutrients that we look at changes with vitamin E supplementation, except a 10% increase in the plasma level of zinc. We’re not sure what sort of change this is in the distribution of the zinc. We don’t know why vitamin E would cause an increase in the plasma level of zinc. Unfortunately, we didn’t look at, for example, the red blood cell or white blood cell level of zinc to see whether there was a decrease in the level of zinc in the membrane. None of the other nutrients, however, showed a change.