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Short-term vitamin E intake fails to improve cognitive or psychomotor performance of aged mice
Free Radical Biology & Medicine, Vol. 36, No. 11, pp. 1424 – 1433, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.02.081
Original Contribution SHORT-TERM VITAMIN E INTAKE FAILS TO IMPROVE COGNITIVE OR PSYCHOMOTOR PERFORMANCE OF AGED MICE NATHALIE SUMIEN,* KEVIN R. HEINRICH,* RAJINDAR S. SOHAL, y and MICHAEL J. FORSTER* *Department of Pharmacology and Neuroscience, Institute for Aging and Alzheimer’s Disease Research, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107 USA; and y Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA 90089, USA (Received 8 December 2003; Revised 26 January 2004; Accepted 27 February 2004) Available online 26 March 2004
Abstract—The purpose of this study was to determine if relatively short-term vitamin E supplementation could reverse age-associated impairments in cognitive or motor function and the accumulated oxidative damage in the brain of aged mice. Separate groups of 5- or 20-month-old C57BL6 mice were placed on either a control diet or the same diet supplemented with a-tocopheryl acetate (1.65 g/kg). After 4 weeks on the diets, mice were tested for cognitive and motor functions over the next 8 weeks, during which the supplementation was maintained. Vitamin E supplementation increased the concentration of a-tocopherol in the cerebral cortex of both the young and old mice, but did not significantly affect oxidative damage to proteins and lipids in the brain cortex. When compared with young controls, the old control mice showed slower learning of a swim maze, longer reaction times, diminished auditory and shock-startle responsiveness, and diminished motor performance on tests of coordinated running and bridge walking. The vitamin E-administered old mice failed to show improvement of function relative to agematched controls on any of the tests, but did show altered retention performance on the swim maze task and impaired performance in the test of coordinated running. The latter effects were not evident in young mice on the supplemented diet. Results of this study suggest that, when implemented in relatively old mice, supplementation of vitamin E is ineffective in reversing preexisting age-related impairments of cognitive or motor function, and has little effect on common measures of protein or lipid oxidative damage in the mouse brain. Moreover, the current findings indicate that vitamin E could have detrimental effects on some brain functions when implemented in older animals. D 2004 Elsevier Inc. All rights reserved. Keywords—Vitamin E, Aging, Cognitive function, Motor function, Free radicals
neurodegenerative conditions such as Alzheimer’s disease [1–6]. Indeed, prolonged administration of vitamin E to mice and rats, initiated at the adult stage, has been reported to attenuate age-associated accumulation of macromolecular oxidative damage in some regions of the brain and to retard the accompanying declines of cognitive and motor performance [7]. Whether or not ameliorative effects can also accrue if vitamin E administration is initiated at a relatively old age is presently unknown. Furthermore, it remains to be determined whether vitamin E may also potentially cause deleterious alterations in brain functions, irrespective of the age at which supplementation is first initiated. This issue is deemed relevant because, based on anticipated health benefits or
INTRODUCTION
Vitamin E, a lipophilic antioxidant, prevents lipid peroxidation by scavenging lipid peroxyl radicals. Its dietary deficiency has been shown to be quite deleterious, especially in the central nervous system; conversely, vitamin E supplementation is reported to have several health benefits, including improvement in mental and physical capacity of healthy aged individuals as well as those suffering from
Address correspondence to: Michael J. Forster, Department of Pharmacology and Neuroscience, UNTHSC, 3500 Camp Bowie, Fort Worth, TX 76107, USA; Fax: 817/735-2091; E-mail: forsterm@hsc. unt.edu. 1424
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on advice from health care professionals, a relatively large number of humans of various ages supplement their diet with exogenous vitamin E. In this context, the purpose of the present study was to determine whether or not vitamin E intake causes any improvement of cognitive or motor performance of mice when implemented at ages subsequent to the appearance of age-related deficits. Accordingly, groups of young (5-month-old) and aged (20-month-old) mice were maintained for 13 weeks on a control diet or a diet supplemented with vitamin E, and tested for their ability to perform on an age-sensitive battery of tests for cognitive and motor function. A swim maze task was employed to measure the ability of the mice to learn and remember the location of a hidden platform. It has been shown that performance on this task is dependent on cortical and hippocampal functions, and is negatively associated with the level of protein oxidative damage [8 – 10]. In addition, a battery of psychomotor tests was used to evaluate different dimensions of age-associated loss, including spontaneous locomotion, coordinated running (rotorod), balance (bridge walking), muscle strength (wire hanging), sensory reactivity (auditory and shock startle responses), and reaction time. MATERIALS AND METHODS
Animals Sixty young (5 months) and sixty old (20 months) male C57BL/6 mice were obtained from the National Institute on Aging and subsequently maintained individually in clear polycarbonate cages (modified into twomouse units with a stainless-steel divider) in the University of North Texas Health Science Center Vivarium. The ambient temperature was maintained at 23 F 1jC, under a 12 h light/dark cycle starting at 0600 h. Mice had ad libitum access to food and water except during the testing sessions. Vitamin E supplementation After a 2-week acclimation period, mice were fed for 13 weeks with either the standard NIH-31 formulation or a formulation to which a-tocopheryl acetate had been added (1.65 g/kg chow). The diets were formulated by Harlan Teklad (Madison, WI, USA) using NIH-31 formula as the base diet (control, TD 96262; supplemented, TD 98119). The acetate ester is hydrolyzed in vivo to the free tocopherol form (activity = 1.0 IU/mg) [11]. Body weights were recorded weekly. Based on the average daily food intake of the mice, the concentration of atocopherol in the diet was designed to deliver a dose of 200 mg/kg body wt/day, similar to that given by gavage in a previous study [10] that resulted in significant
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incorporation of tocopherol into mitochondria and homogenates of various tissues. Testing of the mice for cognitive and psychomotor performance began after 4 weeks of supplementation and continued for the subsequent 8 weeks. One week after completion of behavioral testing, mice were euthanized by carbon dioxide inhalation and the tissues dissected and stored at 80jC. Spatial learning and memory Spatial learning and memory were measured using a swim maze test as described previously[8]. On a given trial, the mouse was allowed to swim in a steel tank (110 cm diam 60 cm deep), filled with opaque water (nontoxic white paint) maintained at 24 F 1jC; an escape was provided by means of a small platform hidden from view 1.5 cm below the surface of the water. A computerized tracking system recorded the length of the path taken by the mouse to reach the platform as well as the swimming speed (San Diego Instruments, San Diego CA, Model SA-3). During the pretraining phase, mice learned the motor components of swimming and climbing onto the platform, without learning its location in the tank. Subsequently, mice were tested for their ability to learn the location of the platform during three phases: acquisition (8 sessions), retention (2 sessions after a 2-day rest), and reversal (4 sessions with the platform at a different location). Each session consisted of five trials during which the mouse had to swim to the platform from a different starting point in the tank. Measurement of locomotor activity and motor skills Locomotor activity. Spontaneous locomotor activity was measured using a Digiscan apparatus (Omnitech Electronics, Model RXYZCM (16)), as described previously [12]. Each mouse was placed in a clear acrylic chamber (40.5 40.5 30.5 cm) for a series of four consecutive time periods, each of 4 min duration. The chamber was surrounded by a metal frame lined with photocells, inside a sound-attenuating chamber equipped with a fan that provided background noise (80 dB). Forward locomotion in centimeters and rearing (movement in a vertical plane 7.6 cm above the floor) were detected by the photocells and recorded by a computer. Walking initiation. This measurement was the average latency (s) to initiate a movement equivalent to the body length after the mouse was placed on a flat smooth surface during each of four consecutive daily sessions. Alley turning. This test determined the average latency (s) to reverse direction when the mouse was placed in
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a dead-end alley on each of four consecutive daily sessions. Negative geotaxis. With the mouse initially facing downward on a flat surface tilted at 45j, the latency (s) to turn 180j facing upward was measured and averaged over the four consecutive daily sessions. Wire suspension. The mouse was allowed to grip a horizontal wire suspended 40 cm above a padded surface, and the latency (s) to fall was recorded and averaged over four consecutive daily sessions. Bridge walking. Each mouse was tested for the latency (s) to fall after being placed on one of four bridges, mounted 45 cm above a padded surface. The bridges differed in diameter (small vs. large) and shape (round or square), providing four levels of difficulty. Each bridge was presented three times and the measure of performance was the average latency to fall (up to a maximum of 60 s) across all bridges. Coordinated running. Motor learning and maximum running performance were measured using an accelerating rotorod test, described previously [13]. The apparatus was a motor-driven treadmill (Omnitech Electronics, Omnirotor treadmill, Model RRF) that consisted of a 3.2 cm diameter nylon cylinder mounted horizontally at a height of 35.5 cm above a padded surface. In a given trial, the mouse was placed on the cylinder, which then began rotating with increasing speed until the animal fell to a well-padded surface. Ability of the mice to improve running performance was considered by administering intermittent training sessions (two per day), each consisting of four trials. The training sessions continued until the running performance (the average latency to fall from the cylinder) failed to show improvement over three consecutive sessions. The treatment and age groups were compared for their average latency to fall on the first seven sessions (completed by all mice in the study) and for the final three sessions on which the mice had reached their maximum level of performance. Sensory reactivity The musculoskeletal startle reflex response to auditory or shock stimuli of various intensities was determined while the mice were restrained in an acrylic cylinder using a standard testing system (SA Lab, San Diego Instruments, San Diego, CA, USA) employing an electromagnetic transducer. For the auditory startle reflex, six different sound intensities (0, 90, 100, 110, 120, and 140 db) lasting 20 ms were emitted 12 times in a counterbalanced series, for a total of 72 trials.
Constant-current shock stimuli (0, 0.02, 0.04, 0.08, 0.12, 0.16, 0.24, 0.32, and 0.64 mA), 100 ms in duration and scrambled across eight inputs to the grid floor of the acrylic chamber, were given five times for a total of 45 trials. The magnitude of the auditory and shock startle responses was considered as the average peak force for each intensity within a 300-ms response window after the stimulus presentation. Reaction time was the average time to peak response after five shock stimuli of maximal intensity (0.64 mA). Biochemical measurements Extraction and quantification of a-tocopherol. Aliquots of 20 –100 Al of tissue homogenate were added to 10 Al EDTA and 750 Al hexane:ethanol (5:2), vortexed for 30 s, and centrifuged at 4000g for 3 min. A 400 Al aliquot of the hexane layer was dried under helium and dissolved in 100 Al of ethanol. Samples were chromatographed on a reverse phase C18 HPLC column (25.0 0.46 cm, 5 Am, Supleco, Inc., Bellefonte, PA, USA) and a mobile phase consisting of 0.7% NaClO4 in ethanol:methanol: 70% HClO4 (900:100:1, v/v/v) at a flow rate of 1.2 ml/min as described by Katayama et al. [14]. The eluant was monitored with an electrochemical detector (guard cell: +200 mV, conditioning cell: 500 mV, analytical cell: +150 mV; ESA Coulochem II, ESA Inc., Bedford, MA, USA). The amount of a-tocopherol was determined by comparison of the peak areas against the standard curve. Measurement of protein oxidative damage. Protein carbonyl concentrations were measured according to the method of Levine et al. [15]. Samples were incubated with either 2, 4-dinitrophenyl hydrazine (DNPH) for the experimentals or HCl for the blanks for 1 h at room temperature in the dark. The difference in absorbance at 366 nm between DNPH-treated and HCl-treated samples was determined and the results were expressed as nanomoles of carbonyls per milligram of protein using an extinction coefficient of 22.0 mM1 cm1. Measurement of lipid oxidative damage. Lipid peroxidation products were measured as thiobarbituric acid-reactive substances (TBARS) as described by Ohkawa et al. [16]. Statistical analysis of data The data from most of the measures were subjected to two-way analyses of variance, with age and supplementation as between-groups factors. Planned individual comparisons of young versus old control groups and between age-matched treatment groups were made using single-degree-of-freedom F tests and the error term for the two-way interaction. Swim maze and coordinated
Vitamin E intake and brain aging
running data were subjected to three-way analyses, with repeated measures on the sessions or the trials factor. RESULTS
Mice from two different age groups, 5 and 20 months, were supplemented with a diet containing a-tocopheryl acetate or maintained on the control diet for a total period of 13 weeks. After 4 weeks of treatment, behavioral tests were initiated in the following order: locomotor activity, motor skills, spatial learning and memory, sensory reactivity. At the end of the 13 weeks, the mice were euthanized and concentrations of a-tocopherol and protein and lipid oxidative damage were measured in the cerebral cortex. Body weight The body weights of the mice in the four groups were recorded weekly throughout the treatment period (Fig. 1). There was no effect of vitamin E intake on the weights of the young or the old mice, although the young groups tended to gain weight whereas the old mice showed weight loss over the treatment period. A three-way analysis of variance on the body weights indicated a significant main effect of age and an age time interaction ( p < .01), but did not show any effect or interaction involving the dietary supplementation. During the study, one old control mouse died prior to behavioral testing and three old supplemented mice died prior to swim maze testing. Spatial learning and memory The length of the path taken to reach the hidden platform (Fig. 2, top) was analyzed to assess the
Fig. 1. Body weight of C57BL/6 mice during the 13 weeks after initiation of a-tocopherol supplementation. Mice were 5 or 20 months of age at the start of the study. Each value represents the mean ( F ) SE of 30 mice.
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efficiency with which the mice located the platform, independently of their speed of swimming (Fig. 2, bottom). Both young and old mice learned to locate the hidden platform efficiently by the 8th session of the acquisition (learning) phase and maintained this level of performance during the retention phase (sessions 9 and 10). However, the young mice tended to show more rapid improvement and better performance than the aged mice on sessions 2 –5. These effects were reflected in significant main effects of age and a significant age x test session interaction ( p values < .04) when data for the acquisition phase were analyzed by ANOVA. Vitamin E intake had no effect on performance of either the young or the old mice during the acquisition or retention phases, as indicated by the absence of significant main effects or interactions involving this factor ( p values > .33). During the first session of the reversal phase, for which the platform was moved to a new position, the aged supplemented mice tended to swim to the new platform position more efficiently than any of the other groups. This effect was reflected in a significant three-way interaction of supplementation, age, and test session ( p < .002). Moreover, individual comparisons confirmed a significant difference between the aged supplemented group and each of the three other groups on session 11 ( p < .012). To determine whether or not this difference reflected faster learning of the platform position by the supplemented group, the performance of the mice was analyzed on individual trials (Fig. 3). This analysis suggested a difference in performance of the old supplemented groups, but did not indicate a difference in learning of the new platform position. The old supplemented group had shorter path lengths than did the old controls on each of the individual trials, although both groups exhibited a similar degree of improvement in performance over the session. The young supplemented and control groups did not differ on any of the trials. This overall pattern resulted in a significant interaction of age and supplementation ( p = .006). There was no significant two- or three-way interaction of trials with supplementation ( p values > .811), although the age trials interaction was significant ( p = .014), reflecting the shorter path lengths of the old as compared with the young groups on the first trial of the session. An analysis of the number of entries into the previous platform position on the first trial of session 11 revealed a significant decline with age ( p < .001), suggesting that the old mice began reversal training with a weaker bias for the previous platform position when compared with the young mice. The effect of supplementation on swimming speed was also age-dependent. The young supplemented groups swam faster than the young controls throughout the three
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Fig. 2. Effects of a-tocopherol administration on swim maze performance as a function of age (left to right). Top: Path length (cm F SE). Bottom: Path-independent swim speed (cm/s F SE) of the mice; *Significant difference from age-matched control (individual comparison within three-way ANOVA).
phases of training, whereas the old vitamin E-supplemented mice tended to swim more slowly than their agematched controls. Analyses of the swimming speed data
revealed a significant interaction between age and supplementation ( p = .012) for the acquisition testing phase, confirming this observation.
Fig. 3. Effects of a-tocopherol supplementation on swim maze performance for the first five trials of the reversal phase as a function of age (left to right). Each panel shows the mean path length (cm F SE) on each of the five individual trials during the first session after the position of the hidden platform had been moved (training session 11, Fig. 2).
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Fig. 4. Effects of a-tocopherol supplementation on motor learning and maximum performance as a function of age (left to right). Each panel shows the mean latency to fall from the rotating cylinder in seconds (F SE) on each of the first seven training sessions and over the final three sessions when no further improvements occurred (Max); *Significant difference from age-matched control (individual comparison within three-way ANOVA).
Coordinated running The effects of age and vitamin E intake on motor learning and maximum running performance are shown in Fig. 4. Both the young and the old mice showed significant improvement in performance over the first seven training sessions, although the old mice initially performed more poorly and achieved a lower maximum level of performance when compared with the young mice. Vitamin E administration did not significantly affect performance of the young mice, but resulted in a diminished level of performance during most of the training sessions in the old mice. The maximum running
performance achieved by the old supplemented mice was 20% lower than that of the old controls. An analysis of variance on these data revealed a significant main effect of supplementation (p < .01). Locomotor activity In tests for spontaneous locomotor activity (Fig. 5), the young control groups tended to show more forward movement and more frequent rearing (standing on the hind legs) when compared with the old groups. There was little effect of vitamin E intake on forward movement in either age group, whereas supplementation
Fig. 5. Effects of a-tocopherol supplementation on spontaneous locomotor activity as a function of age. Left: Forward movement in centimeters (F SE) within the activity chamber. Right: rearing behavior (standing on the hindlimbs), measured by photocell counts (F SE) within a vertical plane 7.6 cm above the floor of the activity chamber. *Significant difference from age-matched control (individual comparison within two-way ANOVA).
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Table 1. Effects of Age and Vitamin E Supplementation on Motor Skills and Sensory Reactivitya Young Control Motor skillsb Walking 2.4 initiation Alley turning 11.6 Negative 9.5 geotaxis Wire grip 35.3 Bridge 45.4 walking Sensory reactivity Auditory startle 4.9 (force units) Shock startle 25.5 (force units) Reaction time 45.7 (ms)
F 0.2
Old VE
Control
VE
3.4 F 0.4
3.1 F 0.3
3.6 F 0.5
F 1.2 11.6 F 0.9 18.8 F 2.2* 17.6 F 2.4 F 0.6 10.9 F 0.9 9.5 F 1.0 9.6 F 1.0 F 2.3 33.8 F 2.7 26.9 F 2.8 28.9 F 2.8 F 2.0 41.0 F 2.9 29.5 F 2.7* 28.9 F 2.8 F 0.7
6.0 F 0.7
1.9 F 0.2*
2.0 F 0.3
F 1.9 25.6 F 1.9 15.5 F 1.3* 16.3 F 1.7 F 2.5 44.1 F 1.7 58.7 F 2.6* 59.2 F 2.6
a
All values are the group means F SE. Mean latency in seconds. * p < .05 when compared with young control group (individual comparison within two-way ANOVA).
main effects of age ( p < .001), reflecting age-related declines in performance, but no apparent effects or interactions involving vitamin E supplementation. There were no significant effects of either age or supplementation on the latency to initiate walking or to turn in a direction opposite to gravity (negative geotaxis). The startle responses of the aged mice to auditory stimuli were markedly diminished at all intensities when compared with the young, as also were the responses of old mice to shock stimuli at most intensities. Analyses of the startle response to maximal shock (0.64 mA) and auditory (140 dB) stimuli indicated significant effects of age ( p < .001), but did not suggest any effect of supplementation (all p > .1). Similarly, age accounted for a 15 ms increase in reaction time ( p < .001), while the supplementation was without effect in both the young and the old mice ( p > .1). a-Tocopherol concentration and oxidative damage
b
resulted in increased rearing activity, most markedly in the young mice. Analyses of the rearing data indicated significant effects of age (p < .016) and supplementation (p < .008), whereas no significant effects were found for forward movement. Motor skills and sensory reactivity Vitamin E intake did not significantly affect performance on several tests for motor skills and sensory reactivity, although significant effects of age were noted (Table 1). Analyses of variance on alley turning, wire grip, and bridge walking latency indicated significant
The concentration of a-tocopherol was increased approximately 3-fold in the plasma of the young and old supplemented mice (Fig. 6, top right) and was increased by 50 to 60% in the cerebral cortex of the same animals (Fig. 6, top left). An ANOVA as well as individual comparisons between age-matched groups revealed a significant effect of supplementation in both the young and old mice ( p < .023). Protein carbonyls and TBARS were measured in the cerebral cortex of young and old supplemented and control mice (Fig. 6, bottom). The carbonyls showed a 1-fold increase with age in the cortex of the mice ( p = .014, main effect), but supplementation did not produce a significant reversal of that effect ( p > .10 for main and interaction effects). Individual comparisons confirmed a
Fig. 6. Effects of a-tocopherol intake on its endogenous concentrations in plasma and brain cortex (top) and amounts of protein carbonyls and TBARS in brain cortex (bottom) of young and old C57BL/6 mice. Values represent means (F SE) of four or five samples, each obtained from a different mouse. *Significant difference from age-matched control. **Significant difference from young control (individual comparison within two-way ANOVA).
Vitamin E intake and brain aging
significant difference between young and old controls ( p = .01), but did not indicate a difference between the old supplemented groups and the young controls ( p > .14). However, the old control and old supplemented groups were not significantly different ( p = .187). There was no significant effect of supplementation or age on the concentration of TBARS in the cortex of these mice ( p > .10 for all effects). DISCUSSION
The main findings of this study are that a-tocopherol intake, when initiated in old mice (a) has little or no effect on oxidative damage to proteins and lipids in the brain, (b) does not improve age-impaired cognitive or motor performance, and (c) adversely affects some psychomotor functions. The present results clearly indicate that mice fed control diets exhibit a deleterious effect of age on many different measures of cognitive, psychomotor, and sensory capacity, which accords with the findings of several previous studies [8,12,13]. However, vitamin E intake did not lead to any improvement in performance for any of the age-sensitive tests, even though the mice had been supplemented for up to 10 weeks prior to testing. These results contrast with earlier reports that vitamin E affords some protection against age-related losses of motor function and swim maze performance when supplementation began 8 months prior to testing [7]. Thus, the results of the current study would seem to indicate that vitamin E supplementation is not beneficial unless it is implemented at ages prior to the appearance of losses in functional capacity. An unanticipated finding was that vitamin E intake had a deleterious effect on coordinated running and swimming speed, and produced alterations in cognitive performance on the swim maze task. These effects were evident after vitamin E supplementation in the old mice, but were absent in young mice receiving supplementation for an equivalent period. It is also important to note that some apparently beneficial alterations in behavior occurred in the young mice when they were administered vitamin E, namely, an increase in swim speed and locomotor activity that was not evident in the old supplemented mice. The differences in behavioral effects of vitamin E supplementation in the young and old mice were not related to any difference in the plasma or brain concentration of vitamin E following the supplementation. Taken together, the age dependence of the effects of vitamin E intake on behavior suggest that aging may confer not only a decline in likelihood of beneficial effects, but also an increase in susceptibility to some apparently deleterious consequences of vitamin E.
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Adverse effects of therapeutic vitamin E supplementation have not been widely reported in the human or animal literature, however, it is noteworthy that in elderly patients with Alzheimer’s disease, Sano et al. [5] reported a 3-fold increase in incidence of falls associated with high dose vitamin E supplementation. This result seems consistent with the current studies of old mice, in which vitamin E supplementation resulted in a decreased latency to fall during the test of coordinated running. The deleterious consequences of vitamin E supplementation in the old mice were restricted to rapid, coordinated activities such as swimming and running, but were not evident on other tests of simple reflexive and motor skills, muscle strength, sensory reactivity, or reaction time. Thus, the adverse consequences of vitamin E supplementation do not seem to involve a generalized deficit in sensory or psychomotor function of the animals. Because vitamin E intake was initiated in relatively old mice in the current studies, our results do not directly address the issue of whether or not vitamin E intake can provide protection against the age-related accumulation of oxidative damage. Indeed, protective effects on oxidative damage have been reported in studies in which vitamin E supplementation is initiated in young or middle-aged subjects and maintained until old age [1,7,17], and these effects are associated with some improvement in cognitive and motor performance. The absence of similar effects on oxidation and behavioral performance in the current studies suggests that vitamin E supplementation affords little benefit when its implementation is delayed until older ages, after increases in oxidative damage have occurred. In this regard, the current negative findings are consistent with the hypothesized relationship between oxidative damage and ageassociated impairment of cognitive and psychomotor function [8,18]. Previous studies have indicated that oxidative damage can be attenuated relatively rapidly in aged animals after implementation of experimental interventions such as restriction of caloric intake [19,20] and administration of certain antioxidant compounds [21,22]. The decrease in oxidative damage produced under these conditions has been associated with improvement in cognitive and/or psychomotor functions [13,22]. It was expected that similar effects might occur after vitamin E supplementation, based on its ability to downregulate mitochondrial oxidant production and oxidative damage in vitro [23], and after short-term supplementation in younger animals [24]. However, the intake of vitamin E failed to significantly affect age-associated oxidative damage in the brains of mice in the current study, and in a previous investigation it did not affect oxidative damage in several peripheral tissues and their mitochondria [25].
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The trend toward decreased protein oxidation in the old supplemented mice in this study could indicate that further increments in availability of vitamin E would result in significant effects on oxidative damage. It has been reported that a relatively smaller accumulation of vitamin E occurs in brain when compared with peripheral tissues after supplementation [26,27] and, based partly on this concern, a relatively high level of supplementation has been used in many clinical trials and animal studies for which brain is the therapeutic target [28]. The current regimen of vitamin E supplementation produced a significant but modest (50 – 60%) increase in the concentration of a-tocopherol in homogenates of the brain cortex, whereas the plasma concentration was increased more than 3-fold. Previous studies have yielded similar small increases in whole brain synaptosomal a-tocopherol concentration after an equivalent supplementation regimen [29]. On the other hand, the current level of supplementation was sufficient to prevent age-dependent losses of cognitive function in apolipoprotein E-deficient mice [30] and falls within the same range as used in another study reporting a similar beneficial effect [17]. The level of vitamin E supplementation used in the current study is also comparable to or higher than that reported to reduce hydrogen peroxide generation [31] and to enhance striatal dopamine release [26] in young animals. Other interpretations of the lack of effect of vitamin E on oxidative damage include the possibility that, being putatively excessive in amount in supplemented animals, oxidized a-tocopherol may not be re-reduced via recycling mechanisms or that extra amounts of a-tocopherol may promote oxidant generation by donating reducing equivalents to redox cycling substances [32], thereby contributing to rather than attenuating oxidative stress. A possible interpretation of the lack of effect on lipid oxidation in this study involves the failure to observe an effect of the age of the mice. This outcome could reflect an insensitivity of the TBARS measure that is also reflected in the absence of an effect of vitamin E supplementation. In other studies, however, the TBARS assay has produced results that parallel those of other measures of oxidative damage [33]. Moreover, some studies have also failed to report age-related increases in markers of brain lipid oxidative damage [34,35]. The adverse effects of vitamin E on some of the behavioral functions in the old mice, in the absence of obvious effects on oxidative damage, may be due to its involvement in regulation of cellular activities by mechanisms unrelated to its antioxidant functions, as described by Azzi and his associates [36 – 38]. For example, vitamin E has been shown to have inhibitory effects on protein kinase C activity, vascular smooth muscle growth, nitric oxide production in endothelial cells, and superoxide
anion radical production in neutrophils and macrophages, among others. Whether such effects are collectively deleterious to the optimal functional capacity of certain organ systems is presently unknown, but remains an important question. To conclude, while the intake of vitamin E may afford protection against certain types of brain insult and may retard progression of some neurodegenerative diseases, the current findings suggest that vitamin E intake may have little or no immediate beneficial impact on the diminished level of function present in healthy aged mice. Given the lack of potential for benefit in this context, it is particularly noteworthy that adverse consequences of vitamin E intake were observed in the present study, and age seems to confer susceptibility to such an effect. The mechanistic basis and broader relevance of the deleterious effects require further exploration. Acknowledgments — This research was supported by Grant RO1 AG17526 from the National Institute on Aging, National Institutes of Health. REFERENCES [1] Casadesus, G.; Shukitt-Hale, B.; Joseph, J. A. Qualitative versus quantitative caloric intake: are they equivalent paths to successful aging? Neurobiol. Aging 23:747 – 769; 2002. [2] La Rue, A.; Koehler, K. M.; Wayne, S. J.; Chiulli, S. J.; Haaland, K. Y.; Garry, P. J. Nutritional status and cognitive functioning in a normally aging sample: a 6-y reassessment. Am. J. Clin. Nutr. 65:20 – 29; 1997. [3] Meydani, M. Nutrition interventions in aging and age-associated disease. Ann. NY Acad. Sci. 928:226 – 235; 2001. [4] Meydani, M. Antioxidants and cognitive function. Nutr. Rev. 59:S75 – 80; discussion S80-72; 2001. [5] Sano, M.; Ernesto, C.; Thomas, R. G.; Klauber, M. R.; Schafer, K. A. A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer’s disease: the Alzheimer’s Disease Cooperative Study. N. Engl. J. Med. 336:1216 – 1222; 1997. [6] Sram, R. J.; Binkova, B.; Topinka, J.; Kotesovec, F.; Fojtikova, I.; Hanel, I.; Klaschka, J.; Kocisova, J.; Prosek, M.; Machalek, J. Effect of antioxidant supplementation in an elderly population. Basic Life Sci. 61:459 – 477; 1993. [7] Joseph, J. A.; Shukitt-Hale, B.; Denisova, N. A.; Prior, R. L.; Cao, G.; Martin, A.; Taglialatela, G.; Bickford, P. C. Long-term dietary strawberry, spinach, or vitamin E supplementation retards the onset of age-related neuronal signal-transduction and cognitive behavioral deficits. J. Neurosci. 18:8047 – 8055; 1998. [8] Forster, M. J.; Dubey, A.; Dawson, K. M.; Stutts, W. A.; Lal, H.; Sohal, R. S. Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain. Proc. Natl. Acad. Sci. USA 93:4765 – 4769; 1996. [9] Nicolle, M. M.; Gonzalez, J.; Sugaya, K.; Baskerville, K. A.; Bryan, D.; Lund, K.; Gallagher, M.; McKinney, M. Signatures of hippocampal oxidative stress in aged spatial learning-impaired rodents. Neuroscience 107:415 – 431; 2001. [10] Lass, A.; Forster, M. J.; Sohal, R. S. Effects of coenzyme Q10 and alpha-tocopehrol administration on their tissue levels in the mouse: elevation of mitochondrial alpha-tocopherol by coenzyme Q10. Free Radic. Biol. Med. 26:1375 – 1382; 1999. [11] Kappus, H.; Diplock, A. T. Tolerance and safety of vitamin E: a toxicological position report. Free Radic. Biol. Med. 13:55 – 74; 1992. [12] Forster, M. J.; Lal, H. Neurobehavioral biomarkers of aging: influence of genotype and dietary restriction. Biomed. Environ. Sci. 4:144 – 165; 1991.
Vitamin E intake and brain aging [13] Forster, M. J.; Lal, H. Estimating age-related changes in psychomotor function: influence of practice and of level of caloric intake in different genotypes. Neurobiol. Aging 20:167 – 176; 1999. [14] Katayama, K.; Takada, M.; Yuzuriha, T.; Abe, K.; Ikenoya, S. Simultaneous determination of ubiquinone-10 and ubiquinol-10 in tissues and mitochondria by high performance liquid chromatography. Biochem. Biophys. Res. Commun. 95:971 – 977; 1980. [15] Levine, R. L.; Williams, J. A.; Stadtman, E. R.; Shacter, E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. 233:346 – 357; 1994. [16] Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351 – 358; 1979. [17] Veinbergs, I.; Mallory, M.; Sagara, Y.; Masliah, E. Vitamin E supplementation prevents spatial learning deficits and dendritic alterations in aged apolipoprotein E-deficient mice. Eur. J. Neurosci. 12:4541 – 4546; 2000. [18] Sohal, R. S.; Forster, M. J. Oxidative stress and senescent decline of brain function. In: Marwah, J.; Teitelbaum, H., editors. Advances in neurodegenerative disorders, Vol. 2: Alzheimer’s and aging. Scottsdale, AZ: Prominent Press; 1998:23 – 48. [19] Dubey, A.; Forster, M. J.; Lal, H.; Sohal, R. S. Effect of age and caloric intake on protein oxidation in different brain regions and on behavioral functions of the mouse. Arch. Biochem. Biophys. 333:189 – 197; 1996. [20] Forster, M. J.; Sohal, B. H.; Sohal, R. S. Reversible effects of long-term caloric restriction on protein oxidative damage. J. Gerontol. A 55:B522 – B529; 2000. [21] Dubey, A.; Forster, M. J.; Sohal, R. S. Effect of the spin-trapping compound N-tert-butyl a-phenylnitrone on protein oxidation and life span. Arch. Biochem. Biophys. 324:249 – 254; 1995. [22] Carney, J. M.; Starke-Reed, P. E.; Oliver, C. N.; Landum, R. W.; Cheng, M. S.; Wu, J. F.; Flyod, R. A. Reversal of age-related increase in brain protein oxidation, decrease in enzyme activity, and loss in temporal and spatial memory by chronic administration of the spin trapping compound N-tert-butyl-a-phenylnitrone. Proc. Natl. Acad. Sci. USA 88:3633 – 3636; 1991. [23] Lass, A.; Sohal, R. S. Effect of coenzyme Q10 and alpha-tocopherol content of mitochondria on the production of superoxide anion radicals. FASEB J. 14:87 – 94; 2000. [24] Chow, C. K. Vitamin E regulation of mitochondrial superoxide generation. Biol. Signals Recept. 10:112 – 124; 2001. [25] Sumien, N.; Forster, M. J.; Sohal, R. S. Supplementation with
[26]
[27]
[28] [29] [30] [31] [32] [33]
[34] [35]
[36] [37] [38]
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vitamin E fails to attenuate oxidative damage in aged mice. Exp. Gerontol. 38:699 – 704; 2003. Martin, A.; Janigian, D.; Shukitt-Hale, B.; Prior, R. L.; Joseph, J. A. Effect of vitamin E intake on levels of vitamin E and C in the central nervous system and peripheral tissues: implications for health recommendations. Brain Res. 845:50 – 59; 1999. Vatassery, G. T.; Brin, M. F.; Fahn, S.; Kayden, H. J.; Traber, M. G. Effect of high doses of dietary vitamin E on the concentrations of vitamin E in several brain regions, plasma, liver, and adipose tissue of rats. J. Neurochem. 51:621 – 623; 1988. Vatassery, G. T.; Bauer, T.; Dysken, M. High doses of vitamin E in the treatment of disorders of the central nervous system in the aged [see comment]. Am. J. Clin. Nutr. 70:793 – 801; 1999. Lass, A.; Sohal, R. S. Comparisons of coenzyme Q bound to mitochondrial membrane proteins among different mammalian species. Free Radic. Biol. Med. 27:220 – 226; 1999. McDonald, S. R.; Forster, M. J. Lifelong tocopheryl acetate administration ameliorates learning deficits in aging apolipoproteinE deficient mice. Soc. Neurosci. Abstr. 26:530; 2000. Chow, C. K.; Ibrahim, W.; Wei, Z.; Chan, A. C. Vitamin E regulates mitochondrial hydrogen peroxide generation. Free Radic. Biol. Med. 27:580 – 587; 1999. Upston, J. M.; Terentis, A. C.; Stocker, R. Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement. FASEB J. 13:977 – 994; 1999. Lass, A.; Sohal, B. H.; Weindruch, R.; Forster, M. J.; Sohal, R. S. Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria. Free Radic. Biol. Med. 25:1089 – 1097; 1998. Cini, M.; Moretti, A. Studies on lipid peroxidation and protein oxidation in the aging brain. Neurobiol. Aging 16:53 – 57; 1995. Matsugo, S.; Kitagawa, T.; Minami, S.; Esashi, Y.; Oomura, Y.; Tokumaru, S.; Kojo, S.; Matsushima, K.; Sasaki, K. Agedependent changes in lipid peroxide levels in peripheral organs, but not in brain, in senescence-accelerated mice. Neurosci. Lett. 278:105 – 108; 2000. Azzi, A.; Ricciarelli, R.; Zingg, J. M. Non-antioxidant molecular functions of alpha-tocopherol (vitamin E). FEBS Lett. 519:8 – 10; 2002. Flohe, R. B.; Kelly, F. J.; Salonen, J. T.; Neuzil, J.; Zingg, J.-M.; Azzi, A. The European perspective on vitamin E: current knowledge and future research. Am. J. Clin. Nutr. 76:703 – 716; 2002. Ricciarelli, R.; Zingg, J. M.; Azzi, A. Vitamin E: protective role of a Janus molecule. FASEB J. 15:2314 – 2325; 2001.
doi:10.1016/j.freeradbiomed.2004.02.081
Original Contribution SHORT-TERM VITAMIN E INTAKE FAILS TO IMPROVE COGNITIVE OR PSYCHOMOTOR PERFORMANCE OF AGED MICE NATHALIE SUMIEN,* KEVIN R. HEINRICH,* RAJINDAR S. SOHAL, y and MICHAEL J. FORSTER* *Department of Pharmacology and Neuroscience, Institute for Aging and Alzheimer’s Disease Research, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107 USA; and y Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA 90089, USA (Received 8 December 2003; Revised 26 January 2004; Accepted 27 February 2004) Available online 26 March 2004
Abstract—The purpose of this study was to determine if relatively short-term vitamin E supplementation could reverse age-associated impairments in cognitive or motor function and the accumulated oxidative damage in the brain of aged mice. Separate groups of 5- or 20-month-old C57BL6 mice were placed on either a control diet or the same diet supplemented with a-tocopheryl acetate (1.65 g/kg). After 4 weeks on the diets, mice were tested for cognitive and motor functions over the next 8 weeks, during which the supplementation was maintained. Vitamin E supplementation increased the concentration of a-tocopherol in the cerebral cortex of both the young and old mice, but did not significantly affect oxidative damage to proteins and lipids in the brain cortex. When compared with young controls, the old control mice showed slower learning of a swim maze, longer reaction times, diminished auditory and shock-startle responsiveness, and diminished motor performance on tests of coordinated running and bridge walking. The vitamin E-administered old mice failed to show improvement of function relative to agematched controls on any of the tests, but did show altered retention performance on the swim maze task and impaired performance in the test of coordinated running. The latter effects were not evident in young mice on the supplemented diet. Results of this study suggest that, when implemented in relatively old mice, supplementation of vitamin E is ineffective in reversing preexisting age-related impairments of cognitive or motor function, and has little effect on common measures of protein or lipid oxidative damage in the mouse brain. Moreover, the current findings indicate that vitamin E could have detrimental effects on some brain functions when implemented in older animals. D 2004 Elsevier Inc. All rights reserved. Keywords—Vitamin E, Aging, Cognitive function, Motor function, Free radicals
neurodegenerative conditions such as Alzheimer’s disease [1–6]. Indeed, prolonged administration of vitamin E to mice and rats, initiated at the adult stage, has been reported to attenuate age-associated accumulation of macromolecular oxidative damage in some regions of the brain and to retard the accompanying declines of cognitive and motor performance [7]. Whether or not ameliorative effects can also accrue if vitamin E administration is initiated at a relatively old age is presently unknown. Furthermore, it remains to be determined whether vitamin E may also potentially cause deleterious alterations in brain functions, irrespective of the age at which supplementation is first initiated. This issue is deemed relevant because, based on anticipated health benefits or
INTRODUCTION
Vitamin E, a lipophilic antioxidant, prevents lipid peroxidation by scavenging lipid peroxyl radicals. Its dietary deficiency has been shown to be quite deleterious, especially in the central nervous system; conversely, vitamin E supplementation is reported to have several health benefits, including improvement in mental and physical capacity of healthy aged individuals as well as those suffering from
Address correspondence to: Michael J. Forster, Department of Pharmacology and Neuroscience, UNTHSC, 3500 Camp Bowie, Fort Worth, TX 76107, USA; Fax: 817/735-2091; E-mail: forsterm@hsc. unt.edu. 1424
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on advice from health care professionals, a relatively large number of humans of various ages supplement their diet with exogenous vitamin E. In this context, the purpose of the present study was to determine whether or not vitamin E intake causes any improvement of cognitive or motor performance of mice when implemented at ages subsequent to the appearance of age-related deficits. Accordingly, groups of young (5-month-old) and aged (20-month-old) mice were maintained for 13 weeks on a control diet or a diet supplemented with vitamin E, and tested for their ability to perform on an age-sensitive battery of tests for cognitive and motor function. A swim maze task was employed to measure the ability of the mice to learn and remember the location of a hidden platform. It has been shown that performance on this task is dependent on cortical and hippocampal functions, and is negatively associated with the level of protein oxidative damage [8 – 10]. In addition, a battery of psychomotor tests was used to evaluate different dimensions of age-associated loss, including spontaneous locomotion, coordinated running (rotorod), balance (bridge walking), muscle strength (wire hanging), sensory reactivity (auditory and shock startle responses), and reaction time. MATERIALS AND METHODS
Animals Sixty young (5 months) and sixty old (20 months) male C57BL/6 mice were obtained from the National Institute on Aging and subsequently maintained individually in clear polycarbonate cages (modified into twomouse units with a stainless-steel divider) in the University of North Texas Health Science Center Vivarium. The ambient temperature was maintained at 23 F 1jC, under a 12 h light/dark cycle starting at 0600 h. Mice had ad libitum access to food and water except during the testing sessions. Vitamin E supplementation After a 2-week acclimation period, mice were fed for 13 weeks with either the standard NIH-31 formulation or a formulation to which a-tocopheryl acetate had been added (1.65 g/kg chow). The diets were formulated by Harlan Teklad (Madison, WI, USA) using NIH-31 formula as the base diet (control, TD 96262; supplemented, TD 98119). The acetate ester is hydrolyzed in vivo to the free tocopherol form (activity = 1.0 IU/mg) [11]. Body weights were recorded weekly. Based on the average daily food intake of the mice, the concentration of atocopherol in the diet was designed to deliver a dose of 200 mg/kg body wt/day, similar to that given by gavage in a previous study [10] that resulted in significant
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incorporation of tocopherol into mitochondria and homogenates of various tissues. Testing of the mice for cognitive and psychomotor performance began after 4 weeks of supplementation and continued for the subsequent 8 weeks. One week after completion of behavioral testing, mice were euthanized by carbon dioxide inhalation and the tissues dissected and stored at 80jC. Spatial learning and memory Spatial learning and memory were measured using a swim maze test as described previously[8]. On a given trial, the mouse was allowed to swim in a steel tank (110 cm diam 60 cm deep), filled with opaque water (nontoxic white paint) maintained at 24 F 1jC; an escape was provided by means of a small platform hidden from view 1.5 cm below the surface of the water. A computerized tracking system recorded the length of the path taken by the mouse to reach the platform as well as the swimming speed (San Diego Instruments, San Diego CA, Model SA-3). During the pretraining phase, mice learned the motor components of swimming and climbing onto the platform, without learning its location in the tank. Subsequently, mice were tested for their ability to learn the location of the platform during three phases: acquisition (8 sessions), retention (2 sessions after a 2-day rest), and reversal (4 sessions with the platform at a different location). Each session consisted of five trials during which the mouse had to swim to the platform from a different starting point in the tank. Measurement of locomotor activity and motor skills Locomotor activity. Spontaneous locomotor activity was measured using a Digiscan apparatus (Omnitech Electronics, Model RXYZCM (16)), as described previously [12]. Each mouse was placed in a clear acrylic chamber (40.5 40.5 30.5 cm) for a series of four consecutive time periods, each of 4 min duration. The chamber was surrounded by a metal frame lined with photocells, inside a sound-attenuating chamber equipped with a fan that provided background noise (80 dB). Forward locomotion in centimeters and rearing (movement in a vertical plane 7.6 cm above the floor) were detected by the photocells and recorded by a computer. Walking initiation. This measurement was the average latency (s) to initiate a movement equivalent to the body length after the mouse was placed on a flat smooth surface during each of four consecutive daily sessions. Alley turning. This test determined the average latency (s) to reverse direction when the mouse was placed in
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a dead-end alley on each of four consecutive daily sessions. Negative geotaxis. With the mouse initially facing downward on a flat surface tilted at 45j, the latency (s) to turn 180j facing upward was measured and averaged over the four consecutive daily sessions. Wire suspension. The mouse was allowed to grip a horizontal wire suspended 40 cm above a padded surface, and the latency (s) to fall was recorded and averaged over four consecutive daily sessions. Bridge walking. Each mouse was tested for the latency (s) to fall after being placed on one of four bridges, mounted 45 cm above a padded surface. The bridges differed in diameter (small vs. large) and shape (round or square), providing four levels of difficulty. Each bridge was presented three times and the measure of performance was the average latency to fall (up to a maximum of 60 s) across all bridges. Coordinated running. Motor learning and maximum running performance were measured using an accelerating rotorod test, described previously [13]. The apparatus was a motor-driven treadmill (Omnitech Electronics, Omnirotor treadmill, Model RRF) that consisted of a 3.2 cm diameter nylon cylinder mounted horizontally at a height of 35.5 cm above a padded surface. In a given trial, the mouse was placed on the cylinder, which then began rotating with increasing speed until the animal fell to a well-padded surface. Ability of the mice to improve running performance was considered by administering intermittent training sessions (two per day), each consisting of four trials. The training sessions continued until the running performance (the average latency to fall from the cylinder) failed to show improvement over three consecutive sessions. The treatment and age groups were compared for their average latency to fall on the first seven sessions (completed by all mice in the study) and for the final three sessions on which the mice had reached their maximum level of performance. Sensory reactivity The musculoskeletal startle reflex response to auditory or shock stimuli of various intensities was determined while the mice were restrained in an acrylic cylinder using a standard testing system (SA Lab, San Diego Instruments, San Diego, CA, USA) employing an electromagnetic transducer. For the auditory startle reflex, six different sound intensities (0, 90, 100, 110, 120, and 140 db) lasting 20 ms were emitted 12 times in a counterbalanced series, for a total of 72 trials.
Constant-current shock stimuli (0, 0.02, 0.04, 0.08, 0.12, 0.16, 0.24, 0.32, and 0.64 mA), 100 ms in duration and scrambled across eight inputs to the grid floor of the acrylic chamber, were given five times for a total of 45 trials. The magnitude of the auditory and shock startle responses was considered as the average peak force for each intensity within a 300-ms response window after the stimulus presentation. Reaction time was the average time to peak response after five shock stimuli of maximal intensity (0.64 mA). Biochemical measurements Extraction and quantification of a-tocopherol. Aliquots of 20 –100 Al of tissue homogenate were added to 10 Al EDTA and 750 Al hexane:ethanol (5:2), vortexed for 30 s, and centrifuged at 4000g for 3 min. A 400 Al aliquot of the hexane layer was dried under helium and dissolved in 100 Al of ethanol. Samples were chromatographed on a reverse phase C18 HPLC column (25.0 0.46 cm, 5 Am, Supleco, Inc., Bellefonte, PA, USA) and a mobile phase consisting of 0.7% NaClO4 in ethanol:methanol: 70% HClO4 (900:100:1, v/v/v) at a flow rate of 1.2 ml/min as described by Katayama et al. [14]. The eluant was monitored with an electrochemical detector (guard cell: +200 mV, conditioning cell: 500 mV, analytical cell: +150 mV; ESA Coulochem II, ESA Inc., Bedford, MA, USA). The amount of a-tocopherol was determined by comparison of the peak areas against the standard curve. Measurement of protein oxidative damage. Protein carbonyl concentrations were measured according to the method of Levine et al. [15]. Samples were incubated with either 2, 4-dinitrophenyl hydrazine (DNPH) for the experimentals or HCl for the blanks for 1 h at room temperature in the dark. The difference in absorbance at 366 nm between DNPH-treated and HCl-treated samples was determined and the results were expressed as nanomoles of carbonyls per milligram of protein using an extinction coefficient of 22.0 mM1 cm1. Measurement of lipid oxidative damage. Lipid peroxidation products were measured as thiobarbituric acid-reactive substances (TBARS) as described by Ohkawa et al. [16]. Statistical analysis of data The data from most of the measures were subjected to two-way analyses of variance, with age and supplementation as between-groups factors. Planned individual comparisons of young versus old control groups and between age-matched treatment groups were made using single-degree-of-freedom F tests and the error term for the two-way interaction. Swim maze and coordinated
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running data were subjected to three-way analyses, with repeated measures on the sessions or the trials factor. RESULTS
Mice from two different age groups, 5 and 20 months, were supplemented with a diet containing a-tocopheryl acetate or maintained on the control diet for a total period of 13 weeks. After 4 weeks of treatment, behavioral tests were initiated in the following order: locomotor activity, motor skills, spatial learning and memory, sensory reactivity. At the end of the 13 weeks, the mice were euthanized and concentrations of a-tocopherol and protein and lipid oxidative damage were measured in the cerebral cortex. Body weight The body weights of the mice in the four groups were recorded weekly throughout the treatment period (Fig. 1). There was no effect of vitamin E intake on the weights of the young or the old mice, although the young groups tended to gain weight whereas the old mice showed weight loss over the treatment period. A three-way analysis of variance on the body weights indicated a significant main effect of age and an age time interaction ( p < .01), but did not show any effect or interaction involving the dietary supplementation. During the study, one old control mouse died prior to behavioral testing and three old supplemented mice died prior to swim maze testing. Spatial learning and memory The length of the path taken to reach the hidden platform (Fig. 2, top) was analyzed to assess the
Fig. 1. Body weight of C57BL/6 mice during the 13 weeks after initiation of a-tocopherol supplementation. Mice were 5 or 20 months of age at the start of the study. Each value represents the mean ( F ) SE of 30 mice.
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efficiency with which the mice located the platform, independently of their speed of swimming (Fig. 2, bottom). Both young and old mice learned to locate the hidden platform efficiently by the 8th session of the acquisition (learning) phase and maintained this level of performance during the retention phase (sessions 9 and 10). However, the young mice tended to show more rapid improvement and better performance than the aged mice on sessions 2 –5. These effects were reflected in significant main effects of age and a significant age x test session interaction ( p values < .04) when data for the acquisition phase were analyzed by ANOVA. Vitamin E intake had no effect on performance of either the young or the old mice during the acquisition or retention phases, as indicated by the absence of significant main effects or interactions involving this factor ( p values > .33). During the first session of the reversal phase, for which the platform was moved to a new position, the aged supplemented mice tended to swim to the new platform position more efficiently than any of the other groups. This effect was reflected in a significant three-way interaction of supplementation, age, and test session ( p < .002). Moreover, individual comparisons confirmed a significant difference between the aged supplemented group and each of the three other groups on session 11 ( p < .012). To determine whether or not this difference reflected faster learning of the platform position by the supplemented group, the performance of the mice was analyzed on individual trials (Fig. 3). This analysis suggested a difference in performance of the old supplemented groups, but did not indicate a difference in learning of the new platform position. The old supplemented group had shorter path lengths than did the old controls on each of the individual trials, although both groups exhibited a similar degree of improvement in performance over the session. The young supplemented and control groups did not differ on any of the trials. This overall pattern resulted in a significant interaction of age and supplementation ( p = .006). There was no significant two- or three-way interaction of trials with supplementation ( p values > .811), although the age trials interaction was significant ( p = .014), reflecting the shorter path lengths of the old as compared with the young groups on the first trial of the session. An analysis of the number of entries into the previous platform position on the first trial of session 11 revealed a significant decline with age ( p < .001), suggesting that the old mice began reversal training with a weaker bias for the previous platform position when compared with the young mice. The effect of supplementation on swimming speed was also age-dependent. The young supplemented groups swam faster than the young controls throughout the three
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Fig. 2. Effects of a-tocopherol administration on swim maze performance as a function of age (left to right). Top: Path length (cm F SE). Bottom: Path-independent swim speed (cm/s F SE) of the mice; *Significant difference from age-matched control (individual comparison within three-way ANOVA).
phases of training, whereas the old vitamin E-supplemented mice tended to swim more slowly than their agematched controls. Analyses of the swimming speed data
revealed a significant interaction between age and supplementation ( p = .012) for the acquisition testing phase, confirming this observation.
Fig. 3. Effects of a-tocopherol supplementation on swim maze performance for the first five trials of the reversal phase as a function of age (left to right). Each panel shows the mean path length (cm F SE) on each of the five individual trials during the first session after the position of the hidden platform had been moved (training session 11, Fig. 2).
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Fig. 4. Effects of a-tocopherol supplementation on motor learning and maximum performance as a function of age (left to right). Each panel shows the mean latency to fall from the rotating cylinder in seconds (F SE) on each of the first seven training sessions and over the final three sessions when no further improvements occurred (Max); *Significant difference from age-matched control (individual comparison within three-way ANOVA).
Coordinated running The effects of age and vitamin E intake on motor learning and maximum running performance are shown in Fig. 4. Both the young and the old mice showed significant improvement in performance over the first seven training sessions, although the old mice initially performed more poorly and achieved a lower maximum level of performance when compared with the young mice. Vitamin E administration did not significantly affect performance of the young mice, but resulted in a diminished level of performance during most of the training sessions in the old mice. The maximum running
performance achieved by the old supplemented mice was 20% lower than that of the old controls. An analysis of variance on these data revealed a significant main effect of supplementation (p < .01). Locomotor activity In tests for spontaneous locomotor activity (Fig. 5), the young control groups tended to show more forward movement and more frequent rearing (standing on the hind legs) when compared with the old groups. There was little effect of vitamin E intake on forward movement in either age group, whereas supplementation
Fig. 5. Effects of a-tocopherol supplementation on spontaneous locomotor activity as a function of age. Left: Forward movement in centimeters (F SE) within the activity chamber. Right: rearing behavior (standing on the hindlimbs), measured by photocell counts (F SE) within a vertical plane 7.6 cm above the floor of the activity chamber. *Significant difference from age-matched control (individual comparison within two-way ANOVA).
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Table 1. Effects of Age and Vitamin E Supplementation on Motor Skills and Sensory Reactivitya Young Control Motor skillsb Walking 2.4 initiation Alley turning 11.6 Negative 9.5 geotaxis Wire grip 35.3 Bridge 45.4 walking Sensory reactivity Auditory startle 4.9 (force units) Shock startle 25.5 (force units) Reaction time 45.7 (ms)
F 0.2
Old VE
Control
VE
3.4 F 0.4
3.1 F 0.3
3.6 F 0.5
F 1.2 11.6 F 0.9 18.8 F 2.2* 17.6 F 2.4 F 0.6 10.9 F 0.9 9.5 F 1.0 9.6 F 1.0 F 2.3 33.8 F 2.7 26.9 F 2.8 28.9 F 2.8 F 2.0 41.0 F 2.9 29.5 F 2.7* 28.9 F 2.8 F 0.7
6.0 F 0.7
1.9 F 0.2*
2.0 F 0.3
F 1.9 25.6 F 1.9 15.5 F 1.3* 16.3 F 1.7 F 2.5 44.1 F 1.7 58.7 F 2.6* 59.2 F 2.6
a
All values are the group means F SE. Mean latency in seconds. * p < .05 when compared with young control group (individual comparison within two-way ANOVA).
main effects of age ( p < .001), reflecting age-related declines in performance, but no apparent effects or interactions involving vitamin E supplementation. There were no significant effects of either age or supplementation on the latency to initiate walking or to turn in a direction opposite to gravity (negative geotaxis). The startle responses of the aged mice to auditory stimuli were markedly diminished at all intensities when compared with the young, as also were the responses of old mice to shock stimuli at most intensities. Analyses of the startle response to maximal shock (0.64 mA) and auditory (140 dB) stimuli indicated significant effects of age ( p < .001), but did not suggest any effect of supplementation (all p > .1). Similarly, age accounted for a 15 ms increase in reaction time ( p < .001), while the supplementation was without effect in both the young and the old mice ( p > .1). a-Tocopherol concentration and oxidative damage
b
resulted in increased rearing activity, most markedly in the young mice. Analyses of the rearing data indicated significant effects of age (p < .016) and supplementation (p < .008), whereas no significant effects were found for forward movement. Motor skills and sensory reactivity Vitamin E intake did not significantly affect performance on several tests for motor skills and sensory reactivity, although significant effects of age were noted (Table 1). Analyses of variance on alley turning, wire grip, and bridge walking latency indicated significant
The concentration of a-tocopherol was increased approximately 3-fold in the plasma of the young and old supplemented mice (Fig. 6, top right) and was increased by 50 to 60% in the cerebral cortex of the same animals (Fig. 6, top left). An ANOVA as well as individual comparisons between age-matched groups revealed a significant effect of supplementation in both the young and old mice ( p < .023). Protein carbonyls and TBARS were measured in the cerebral cortex of young and old supplemented and control mice (Fig. 6, bottom). The carbonyls showed a 1-fold increase with age in the cortex of the mice ( p = .014, main effect), but supplementation did not produce a significant reversal of that effect ( p > .10 for main and interaction effects). Individual comparisons confirmed a
Fig. 6. Effects of a-tocopherol intake on its endogenous concentrations in plasma and brain cortex (top) and amounts of protein carbonyls and TBARS in brain cortex (bottom) of young and old C57BL/6 mice. Values represent means (F SE) of four or five samples, each obtained from a different mouse. *Significant difference from age-matched control. **Significant difference from young control (individual comparison within two-way ANOVA).
Vitamin E intake and brain aging
significant difference between young and old controls ( p = .01), but did not indicate a difference between the old supplemented groups and the young controls ( p > .14). However, the old control and old supplemented groups were not significantly different ( p = .187). There was no significant effect of supplementation or age on the concentration of TBARS in the cortex of these mice ( p > .10 for all effects). DISCUSSION
The main findings of this study are that a-tocopherol intake, when initiated in old mice (a) has little or no effect on oxidative damage to proteins and lipids in the brain, (b) does not improve age-impaired cognitive or motor performance, and (c) adversely affects some psychomotor functions. The present results clearly indicate that mice fed control diets exhibit a deleterious effect of age on many different measures of cognitive, psychomotor, and sensory capacity, which accords with the findings of several previous studies [8,12,13]. However, vitamin E intake did not lead to any improvement in performance for any of the age-sensitive tests, even though the mice had been supplemented for up to 10 weeks prior to testing. These results contrast with earlier reports that vitamin E affords some protection against age-related losses of motor function and swim maze performance when supplementation began 8 months prior to testing [7]. Thus, the results of the current study would seem to indicate that vitamin E supplementation is not beneficial unless it is implemented at ages prior to the appearance of losses in functional capacity. An unanticipated finding was that vitamin E intake had a deleterious effect on coordinated running and swimming speed, and produced alterations in cognitive performance on the swim maze task. These effects were evident after vitamin E supplementation in the old mice, but were absent in young mice receiving supplementation for an equivalent period. It is also important to note that some apparently beneficial alterations in behavior occurred in the young mice when they were administered vitamin E, namely, an increase in swim speed and locomotor activity that was not evident in the old supplemented mice. The differences in behavioral effects of vitamin E supplementation in the young and old mice were not related to any difference in the plasma or brain concentration of vitamin E following the supplementation. Taken together, the age dependence of the effects of vitamin E intake on behavior suggest that aging may confer not only a decline in likelihood of beneficial effects, but also an increase in susceptibility to some apparently deleterious consequences of vitamin E.
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Adverse effects of therapeutic vitamin E supplementation have not been widely reported in the human or animal literature, however, it is noteworthy that in elderly patients with Alzheimer’s disease, Sano et al. [5] reported a 3-fold increase in incidence of falls associated with high dose vitamin E supplementation. This result seems consistent with the current studies of old mice, in which vitamin E supplementation resulted in a decreased latency to fall during the test of coordinated running. The deleterious consequences of vitamin E supplementation in the old mice were restricted to rapid, coordinated activities such as swimming and running, but were not evident on other tests of simple reflexive and motor skills, muscle strength, sensory reactivity, or reaction time. Thus, the adverse consequences of vitamin E supplementation do not seem to involve a generalized deficit in sensory or psychomotor function of the animals. Because vitamin E intake was initiated in relatively old mice in the current studies, our results do not directly address the issue of whether or not vitamin E intake can provide protection against the age-related accumulation of oxidative damage. Indeed, protective effects on oxidative damage have been reported in studies in which vitamin E supplementation is initiated in young or middle-aged subjects and maintained until old age [1,7,17], and these effects are associated with some improvement in cognitive and motor performance. The absence of similar effects on oxidation and behavioral performance in the current studies suggests that vitamin E supplementation affords little benefit when its implementation is delayed until older ages, after increases in oxidative damage have occurred. In this regard, the current negative findings are consistent with the hypothesized relationship between oxidative damage and ageassociated impairment of cognitive and psychomotor function [8,18]. Previous studies have indicated that oxidative damage can be attenuated relatively rapidly in aged animals after implementation of experimental interventions such as restriction of caloric intake [19,20] and administration of certain antioxidant compounds [21,22]. The decrease in oxidative damage produced under these conditions has been associated with improvement in cognitive and/or psychomotor functions [13,22]. It was expected that similar effects might occur after vitamin E supplementation, based on its ability to downregulate mitochondrial oxidant production and oxidative damage in vitro [23], and after short-term supplementation in younger animals [24]. However, the intake of vitamin E failed to significantly affect age-associated oxidative damage in the brains of mice in the current study, and in a previous investigation it did not affect oxidative damage in several peripheral tissues and their mitochondria [25].
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The trend toward decreased protein oxidation in the old supplemented mice in this study could indicate that further increments in availability of vitamin E would result in significant effects on oxidative damage. It has been reported that a relatively smaller accumulation of vitamin E occurs in brain when compared with peripheral tissues after supplementation [26,27] and, based partly on this concern, a relatively high level of supplementation has been used in many clinical trials and animal studies for which brain is the therapeutic target [28]. The current regimen of vitamin E supplementation produced a significant but modest (50 – 60%) increase in the concentration of a-tocopherol in homogenates of the brain cortex, whereas the plasma concentration was increased more than 3-fold. Previous studies have yielded similar small increases in whole brain synaptosomal a-tocopherol concentration after an equivalent supplementation regimen [29]. On the other hand, the current level of supplementation was sufficient to prevent age-dependent losses of cognitive function in apolipoprotein E-deficient mice [30] and falls within the same range as used in another study reporting a similar beneficial effect [17]. The level of vitamin E supplementation used in the current study is also comparable to or higher than that reported to reduce hydrogen peroxide generation [31] and to enhance striatal dopamine release [26] in young animals. Other interpretations of the lack of effect of vitamin E on oxidative damage include the possibility that, being putatively excessive in amount in supplemented animals, oxidized a-tocopherol may not be re-reduced via recycling mechanisms or that extra amounts of a-tocopherol may promote oxidant generation by donating reducing equivalents to redox cycling substances [32], thereby contributing to rather than attenuating oxidative stress. A possible interpretation of the lack of effect on lipid oxidation in this study involves the failure to observe an effect of the age of the mice. This outcome could reflect an insensitivity of the TBARS measure that is also reflected in the absence of an effect of vitamin E supplementation. In other studies, however, the TBARS assay has produced results that parallel those of other measures of oxidative damage [33]. Moreover, some studies have also failed to report age-related increases in markers of brain lipid oxidative damage [34,35]. The adverse effects of vitamin E on some of the behavioral functions in the old mice, in the absence of obvious effects on oxidative damage, may be due to its involvement in regulation of cellular activities by mechanisms unrelated to its antioxidant functions, as described by Azzi and his associates [36 – 38]. For example, vitamin E has been shown to have inhibitory effects on protein kinase C activity, vascular smooth muscle growth, nitric oxide production in endothelial cells, and superoxide
anion radical production in neutrophils and macrophages, among others. Whether such effects are collectively deleterious to the optimal functional capacity of certain organ systems is presently unknown, but remains an important question. To conclude, while the intake of vitamin E may afford protection against certain types of brain insult and may retard progression of some neurodegenerative diseases, the current findings suggest that vitamin E intake may have little or no immediate beneficial impact on the diminished level of function present in healthy aged mice. Given the lack of potential for benefit in this context, it is particularly noteworthy that adverse consequences of vitamin E intake were observed in the present study, and age seems to confer susceptibility to such an effect. The mechanistic basis and broader relevance of the deleterious effects require further exploration. Acknowledgments — This research was supported by Grant RO1 AG17526 from the National Institute on Aging, National Institutes of Health. REFERENCES [1] Casadesus, G.; Shukitt-Hale, B.; Joseph, J. A. Qualitative versus quantitative caloric intake: are they equivalent paths to successful aging? Neurobiol. Aging 23:747 – 769; 2002. [2] La Rue, A.; Koehler, K. M.; Wayne, S. J.; Chiulli, S. J.; Haaland, K. Y.; Garry, P. J. Nutritional status and cognitive functioning in a normally aging sample: a 6-y reassessment. Am. J. Clin. Nutr. 65:20 – 29; 1997. [3] Meydani, M. Nutrition interventions in aging and age-associated disease. Ann. NY Acad. Sci. 928:226 – 235; 2001. [4] Meydani, M. Antioxidants and cognitive function. Nutr. Rev. 59:S75 – 80; discussion S80-72; 2001. [5] Sano, M.; Ernesto, C.; Thomas, R. G.; Klauber, M. R.; Schafer, K. A. A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer’s disease: the Alzheimer’s Disease Cooperative Study. N. Engl. J. Med. 336:1216 – 1222; 1997. [6] Sram, R. J.; Binkova, B.; Topinka, J.; Kotesovec, F.; Fojtikova, I.; Hanel, I.; Klaschka, J.; Kocisova, J.; Prosek, M.; Machalek, J. Effect of antioxidant supplementation in an elderly population. Basic Life Sci. 61:459 – 477; 1993. [7] Joseph, J. A.; Shukitt-Hale, B.; Denisova, N. A.; Prior, R. L.; Cao, G.; Martin, A.; Taglialatela, G.; Bickford, P. C. Long-term dietary strawberry, spinach, or vitamin E supplementation retards the onset of age-related neuronal signal-transduction and cognitive behavioral deficits. J. Neurosci. 18:8047 – 8055; 1998. [8] Forster, M. J.; Dubey, A.; Dawson, K. M.; Stutts, W. A.; Lal, H.; Sohal, R. S. Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain. Proc. Natl. Acad. Sci. USA 93:4765 – 4769; 1996. [9] Nicolle, M. M.; Gonzalez, J.; Sugaya, K.; Baskerville, K. A.; Bryan, D.; Lund, K.; Gallagher, M.; McKinney, M. Signatures of hippocampal oxidative stress in aged spatial learning-impaired rodents. Neuroscience 107:415 – 431; 2001. [10] Lass, A.; Forster, M. J.; Sohal, R. S. Effects of coenzyme Q10 and alpha-tocopehrol administration on their tissue levels in the mouse: elevation of mitochondrial alpha-tocopherol by coenzyme Q10. Free Radic. Biol. Med. 26:1375 – 1382; 1999. [11] Kappus, H.; Diplock, A. T. Tolerance and safety of vitamin E: a toxicological position report. Free Radic. Biol. Med. 13:55 – 74; 1992. [12] Forster, M. J.; Lal, H. Neurobehavioral biomarkers of aging: influence of genotype and dietary restriction. Biomed. Environ. Sci. 4:144 – 165; 1991.
Vitamin E intake and brain aging [13] Forster, M. J.; Lal, H. Estimating age-related changes in psychomotor function: influence of practice and of level of caloric intake in different genotypes. Neurobiol. Aging 20:167 – 176; 1999. [14] Katayama, K.; Takada, M.; Yuzuriha, T.; Abe, K.; Ikenoya, S. Simultaneous determination of ubiquinone-10 and ubiquinol-10 in tissues and mitochondria by high performance liquid chromatography. Biochem. Biophys. Res. Commun. 95:971 – 977; 1980. [15] Levine, R. L.; Williams, J. A.; Stadtman, E. R.; Shacter, E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. 233:346 – 357; 1994. [16] Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351 – 358; 1979. [17] Veinbergs, I.; Mallory, M.; Sagara, Y.; Masliah, E. Vitamin E supplementation prevents spatial learning deficits and dendritic alterations in aged apolipoprotein E-deficient mice. Eur. J. Neurosci. 12:4541 – 4546; 2000. [18] Sohal, R. S.; Forster, M. J. Oxidative stress and senescent decline of brain function. In: Marwah, J.; Teitelbaum, H., editors. Advances in neurodegenerative disorders, Vol. 2: Alzheimer’s and aging. Scottsdale, AZ: Prominent Press; 1998:23 – 48. [19] Dubey, A.; Forster, M. J.; Lal, H.; Sohal, R. S. Effect of age and caloric intake on protein oxidation in different brain regions and on behavioral functions of the mouse. Arch. Biochem. Biophys. 333:189 – 197; 1996. [20] Forster, M. J.; Sohal, B. H.; Sohal, R. S. Reversible effects of long-term caloric restriction on protein oxidative damage. J. Gerontol. A 55:B522 – B529; 2000. [21] Dubey, A.; Forster, M. J.; Sohal, R. S. Effect of the spin-trapping compound N-tert-butyl a-phenylnitrone on protein oxidation and life span. Arch. Biochem. Biophys. 324:249 – 254; 1995. [22] Carney, J. M.; Starke-Reed, P. E.; Oliver, C. N.; Landum, R. W.; Cheng, M. S.; Wu, J. F.; Flyod, R. A. Reversal of age-related increase in brain protein oxidation, decrease in enzyme activity, and loss in temporal and spatial memory by chronic administration of the spin trapping compound N-tert-butyl-a-phenylnitrone. Proc. Natl. Acad. Sci. USA 88:3633 – 3636; 1991. [23] Lass, A.; Sohal, R. S. Effect of coenzyme Q10 and alpha-tocopherol content of mitochondria on the production of superoxide anion radicals. FASEB J. 14:87 – 94; 2000. [24] Chow, C. K. Vitamin E regulation of mitochondrial superoxide generation. Biol. Signals Recept. 10:112 – 124; 2001. [25] Sumien, N.; Forster, M. J.; Sohal, R. S. Supplementation with
[26]
[27]
[28] [29] [30] [31] [32] [33]
[34] [35]
[36] [37] [38]
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vitamin E fails to attenuate oxidative damage in aged mice. Exp. Gerontol. 38:699 – 704; 2003. Martin, A.; Janigian, D.; Shukitt-Hale, B.; Prior, R. L.; Joseph, J. A. Effect of vitamin E intake on levels of vitamin E and C in the central nervous system and peripheral tissues: implications for health recommendations. Brain Res. 845:50 – 59; 1999. Vatassery, G. T.; Brin, M. F.; Fahn, S.; Kayden, H. J.; Traber, M. G. Effect of high doses of dietary vitamin E on the concentrations of vitamin E in several brain regions, plasma, liver, and adipose tissue of rats. J. Neurochem. 51:621 – 623; 1988. Vatassery, G. T.; Bauer, T.; Dysken, M. High doses of vitamin E in the treatment of disorders of the central nervous system in the aged [see comment]. Am. J. Clin. Nutr. 70:793 – 801; 1999. Lass, A.; Sohal, R. S. Comparisons of coenzyme Q bound to mitochondrial membrane proteins among different mammalian species. Free Radic. Biol. Med. 27:220 – 226; 1999. McDonald, S. R.; Forster, M. J. Lifelong tocopheryl acetate administration ameliorates learning deficits in aging apolipoproteinE deficient mice. Soc. Neurosci. Abstr. 26:530; 2000. Chow, C. K.; Ibrahim, W.; Wei, Z.; Chan, A. C. Vitamin E regulates mitochondrial hydrogen peroxide generation. Free Radic. Biol. Med. 27:580 – 587; 1999. Upston, J. M.; Terentis, A. C.; Stocker, R. Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement. FASEB J. 13:977 – 994; 1999. Lass, A.; Sohal, B. H.; Weindruch, R.; Forster, M. J.; Sohal, R. S. Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria. Free Radic. Biol. Med. 25:1089 – 1097; 1998. Cini, M.; Moretti, A. Studies on lipid peroxidation and protein oxidation in the aging brain. Neurobiol. Aging 16:53 – 57; 1995. Matsugo, S.; Kitagawa, T.; Minami, S.; Esashi, Y.; Oomura, Y.; Tokumaru, S.; Kojo, S.; Matsushima, K.; Sasaki, K. Agedependent changes in lipid peroxide levels in peripheral organs, but not in brain, in senescence-accelerated mice. Neurosci. Lett. 278:105 – 108; 2000. Azzi, A.; Ricciarelli, R.; Zingg, J. M. Non-antioxidant molecular functions of alpha-tocopherol (vitamin E). FEBS Lett. 519:8 – 10; 2002. Flohe, R. B.; Kelly, F. J.; Salonen, J. T.; Neuzil, J.; Zingg, J.-M.; Azzi, A. The European perspective on vitamin E: current knowledge and future research. Am. J. Clin. Nutr. 76:703 – 716; 2002. Ricciarelli, R.; Zingg, J. M.; Azzi, A. Vitamin E: protective role of a Janus molecule. FASEB J. 15:2314 – 2325; 2001.