Eur J Nutr DOI 10.1007/s00394-013-0628-1
ORIGINAL CONTRIBUTION
The effect of antioxidant vitamins E and C on cognitive performance of the elderly with mild cognitive impairment in Isfahan, Iran: a double-blind, randomized, placebo-controlled trial A. M. Alavi Naeini • I. Elmadfa • A. Djazayery M. Barekatain • M. R. Aghaye Ghazvini • M. Djalali • A. Feizi
•
Received: 15 June 2013 / Accepted: 18 November 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose This study was carried out to investigate the effect of vitamins E and C on cognitive performance among the elderly in Iran. Methods About 256 elderly with mild cognitive impairment, aged 60–75 years, received 300 mg of vitamin E plus 400 mg of vitamin C or placebo daily just for 1 year. Background Demographic characteristics, anthropometric variables food consumption, cognitive function by Mini-Mental State Examination (MMSE), and some of the oxidative stress biomarkers were examined. Results Antioxidant supplementation reduced malondialdehyde level (P \ 0.001) and raised total antioxidant capacity (P \ 0.001) and glutathione (P \ 0.01). The A. M. Alavi Naeini A. Djazayery M. Djalali School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, Iran I. Elmadfa Institute of Nutritional Sciences, University of Vienna, Vienna, Austria I. Elmadfa (&) International Union of Nutritional Sciences (IUNS), Althanstrasse 14, 1090 Vienna, Austria e-mail: [email protected] M. Barekatain Department of Psychiatry and Behavioral Sciences, Isfahan University of Medical Sciences, Isfahan, Iran M. R. Aghaye Ghazvini Isfahan Center of Health Research, Nationally Institute of Health Research, Tehran University of Medical Science, Tehran, Iran A. Feizi Department of Biostatistics and Epidemiology, Isfahan University of Medical Sciences, Isfahan, Iran
serum 8-hydroxydeoxyguanosine remained unchanged (P \ 0.4). After adjusting for the covariates effects, MMSE scores following 6- (25.88 ± 0.17) and 12-month antioxidant supplementation (26.8 ± 0.17) did not differ from control group (25.86 ± 0.18 and 26.59 ± 0.18, respectively). Conclusion Despite significant improvement in most of the oxidative stress biomarkers, antioxidants’ supplementation was not observed to enhance cognitive performance. A large number of kinetic and/or dynamic factors could be suspected. Keywords Supplementation The elderly Mild cognitive impairment Vitamin E Vitamin C
Introduction Dementia is defined as a progressive loss of brain cognitive function that mainly affects older people. It can be occurred by a number of etiologies, which lead to a decline in memory, judgment, decision making, learning, executive functioning, and other mental activities. The symptoms would cause clinically significant distress or impairment in social, occupational, or other important areas of functioning [1]. Alzheimer’s disease, for example, is the most known cause of dementia, characterized by severe cognitive and functional disabilities [2]. As a slowly progressive process, cognitive decline in its early stages may not impair important area of functioning. Mild cognitive impairment (MCI) is another term, which has been defined as a transitional stage between normal cognitive function and dementia. People with MCI have minor problems with memory, attention, speech, decision making, visuospatial, and psychomotor functions, which exceed those expected
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for their age and educational level [3]. However, there is no sign of major impairment in social, occupational, or personal functioning. The patients suffering from MCI have a significantly higher rate, about tenfold, of progression to Alzheimer’s disease compared to cognitively normal elderly people [4, 5]. Despite considerable investigations, the exact factors associated the induction of MCI or its more rapid progression to Alzheimer’s disease has not been well defined. Extensive evidence indicates that oxidative stress plays an important role in the pathogenesis of cognitive disorders. Oxidative stress has been defined as a state where highly reactive free radicals activity exceeds the antioxidant systems defense. This can lead to chemical oxidative modification of macromolecules and change the structure and function of cellular key components [4, 6, 7]. Increased level of oxidative stress markers including malondialdehyde (MDA) [4, 8] and (8-hydroxydeoxyguanosine) 8-OHdG [9] and decreased levels of glutathione (GSH) [10], total antioxidant capacity (TAC) [11], and antioxidant enzymes [4] have been reported in MCI patients. Cerebral tissue is particularly susceptible to reactive oxygen spices (ROS) damage due to its high oxygen requirements for metabolism, low content of antioxidants, and a high content of polyunsaturated fatty acids [4, 6, 7]. These oxidative events have been implicated both in MCI [12] and its progression to Alzheimer’s disease [13]. Regarding these observations, antioxidant vitamins, including vitamins E, the most potent lipophilic chainbreaking antioxidant [14], and vitamin C, a potent watersoluble antioxidant, are expected to reduce neuronal damage and improve cognitive performance of these patients; however, previous studies have yielded conflicting results in this regard. While some clinical and epidemiological studies pointed to the slowing of cognitive decline with the use of vitamins C and/or E [15–19], some others failed to find any protective effect for these antioxidants [20–22]. The aim of the present study was to examine the effects of antioxidant vitamins E and C supplements on cognitive performance in a group of elderly males and females in Iran.
disease, Alcohol intake, smoking, and routine consumption of neurological or antioxidants drugs. An expert psychologist evaluated the subjects to find those suffering from MCI on the basis of attaining 21–26 scores in Mini-Mental State Examination (MMSE). The Iranian version of MMSE had been validated and found to be applicable for the local elderly population [23]. Of the 761 volunteers, 296 were found to have MCI and selected for the next part of the study. From these, 40 did not continue with the study due to the problems tolerating the supplementations (14 subjects), 1 subject died, and 25 subjects abstained to continue participation due to personal reasons. Therefore, 256 subjects completed the study. As shown in Fig. 1, finally selected subjects were randomly assigned into two groups, namely intervention and control. Selection and grouping were performed using stratified method followed by simple randomization method. The present study protocol was conducted in accordance with the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Ethics Committee at Ministry of Health and Medical Education, Iran. To participate in this study, all the subjects or their families signed the consent forms.
Subjects and methods
Data analysis
Subjects and study design
The results are presented as mean ± standard error of mean (SEM) for quantitative variables and number (percent) for qualitative variables. Repeated measures analysis of variance (ANOVA) was used for analyzing the data as the main statistical method. Mauchly’s sphericity test was conducted to assess sphericity as a perquisite assumption.
This study was designed as a double-blind, randomized, controlled clinical trial. From retirees clubs, 761 elderly volunteers with ages between 60 and 75 years were recruited. Exclusion criteria included obvious disabling
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Procedures One group consumed 300 mg of vitamin E (Dl-alphatocopherol acetate) plus 400 mg vitamin C (ascorbic acid) per day, and the second group consumed placebo with the identical condition over the 1-year intervention period. Fasting venous blood from each subject was obtained at three time points, namely on days 0, 180, and 360. Serum MDA was measured according to the method described by Yu et al. [24]. TAC was assessed based on Miller and RiceEvans [25]. Red blood cells GSH was determined quantitatively using PerkinElmer spectrophotometer according to the method of Beutler et al. [26]. 8-OHdG was measured using a competitive enzyme-linked immunosorbent assay (ELISA) kit from Immundiagnostik Company, Germany. Cognitive performance was evaluated by MMSE at three time points similar to blood sampling. Three-day dietary record forms were also completed for each participant by a trained nutritionist every 2 months throughout the study.
Eur J Nutr Fig. 1 Stratified and simple randomization
Table 1 Descriptive characteristics of the subjects at baseline Characteristic
Supplemented (n = 127)
Control (n = 129)
P value*
Age (year) Weight (kg)
66.5 ± 0.39 71.0 ± 0.94
66.3 ± 0.38 69.1 ± 0.94
0.755 0.139 0.703
Height (cm)
162.5 ± 0.78
162 ± 0.86
BMI (kg/m2)
26.9 ± 0.35
26.3 ± 0.35
0.233
Systolic blood pressure
141.2 ± 1.67
137.4 ± 1.7
0.119
Diastolic blood pressure
80.2 ± 1.2
77.9 ± 1.2
0.164
Male n (%)
63 (49.6)
57 (44.2)
0.229
Female n (%)
64 (50.4)
72 (55.8)
25 (19.8)
16 (12.5)
Sex
Results General characteristic of the subjects
Educational level Primary n (%)
When the assumption was not satisfied, Huynh–Feldt correction was used. Within-group comparisons at each follow-up time point were made using repeated contrasts. Between-group comparisons were conducted using two independent samples t test. Chi square test for difference between proportions was used. Analysis of the data was done by SPSS software version 19. Analysis of consumed foods was done by Food Processor II software. For all hypotheses, a significance level of P \ 0.05 was considered statistically significant.
Secondary n (%)
17 (13.5)
10 (7.8)
Diploma n (%)
48 (38.1)
55 (43.0)
University degree n (%)
36 (28.6)
47 (36.7)
* Student’s t test or chi–square test
0.127
General characteristics of the studied subjects for both groups are summarized in Table 1. As can be seen, no significant differences were found between supplemented and control groups (Student’s t test or Chi square tests). Data from 3-day dietary record for intakes of vitamin C, vitamin E, carotene, selenium, and zinc were analyzed (Table 2) to compare the dietary antioxidant
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Eur J Nutr Table 2 Means and standard error of dietary intake antioxidants based on 3-day dietary record every 2 month during 1 year for supplemented and control groups Variable groups Vitamin E (mg)
Vitamin C (mg)
Carotene (mg)
Selenium (lg)
Zinc (mg)
Supplemented
First time
Second time
Third time
Fourth time
Fifth time
Sixth time
12.0 ± 0.9
12.2 ± 1.0
10.7 ± 0.4
11.9 ± 0.9
10.0 ± 0.5
10.4 ± 0.4
Control
11.6 ± 0.8
10.0 ± 0.4
10.4 ± 0.4
10.2 ± 0.4
10.6 ± 0.6
10.2 ± 0.4
P value
0.357
0.134
0.672
0.625
0.433
0.134
Supplemented
90.7 ± 6.4
97.0 ± 6.5
92.6 ± 5.9
118.4 ± 20.1
103.5 ± 6.2
107 ± 5.6
Control
98.6 ± 5.8
110.7 ± 6.4
99.9 ± 5.9
108.4 ± 6.2
117.6 ± 6.8
109 ± 5.4
P value
0.729
0.029*
0.376
0.066
0.126
0.779
Supplemented Control
759 ± 63.6 687 ± 54.2
668 ± 62.1 653 ± 59.1
575 ± 47.6 647 ± 52.5
593 ± 46.5 589 ± 48.6
594 ± 55.0 566 ± 46.5
502 ± 43.4 540 ± 42.5
P value
0.388
0.866
0.306
0.960
0.698
0.525
Supplemented
69.3 ± 4.1
75.6 ± 5.0
78.0 ± 6.0
84.7 ± 6.1
74.6 ± 6.0
70.2 ± 4.1
Control
67.7 ± 4.2
66.9 ± 4.3
72.0 ± 4.8
70.0 ± 4.7
66.3 ± 4.9
74.1 ± 4.9
P value
0.787
0.188
0.415
0.550
0.281
0.552
Supplemented
9.2 ± 0.3
8.9 ± 0.4
8.7 ± 0.3
9.6 ± 0.7
8.4 ± 0.3
8.6 ± 0.3
Control
9.1 ± 0.4
8.6 ± 0.3
9.6 ± 0.7
8.7 ± 0.3
8.4 ± 0.3
8.9 ± 0.3
* Independent t test P \ 0.029
Table 3 Results of within- and between-group comparisons for the MDA, TAC, GSH, and 8-OHdG based on repeated measures ANOVA Variable groups MDA
TAC
GSH
8-OHdG
Period 0
Period 180
Period 360
Time
Treatment
Time 9 treatment
Supplemented
1.54 ± 0.054
2.37 ± 0.055
1.75 ± 0.067
F = 153.7
F = 9.49
F = 2.44
Control
1.56 ± 0.052
2.54 ± 0.055
2.17 ± 0.089
P \ 0.001**
P = 0.002**
P = 0.12
t value
t = 0.27
t = 2.3
t = 3.8
P value
P = 0.78
P = 0.02
P \ 0.001
Supplemented
1.08 ± 0.022
1.47 ± 0.028
1.78 ± 0.048
F = 241.0
F = 16.77
F = 16.38
Control
1.06 ± 0.019
1.43 ± 0.027
1.49 ± 0.031
P \ 0.001**
P \ 0.001**
P \ 0.001**
t value P value
t = -652 P \ 0.51
t = -1.02 P \ 0.31
t = -5.17 P \ 0.001*
Supplemented
61.7 ± 2.84
58.9 ± 1.25
64.5 ± 1.38
F = 1.683
F = 0.236
F = 5.580
Control
59.7 ± 2.27
60.9 ± 1.31
59.7 ± 1.25
P = 0.18
P = 0.63
P = 0.02**
t value
t = -0.529
t = 1.23
t = -2.52
P value
P \ 0.59
P \ 0.22
P \ 0.001*
Supplemented
0.129 ± 0.007
0.097 ± 0.003
0.082 ± 0.003
F = 31.53
F = 0.710
F = 0.976
Control
0.134 ± 0.010
0.097 ± 0.003
0.092 ± 0.007
P \ 0.001**
P = 0.401
P = 0.350
t value
t = -0.359
T = 0.019
t = -1.329
P value
P \ 0.72
P \ 0.98
P \ 0.18
** Repeated measure ANOVA, * Student’s t test
Oxidative stress biomarkers
second half-year of the study (P \ 0.001, repeated measures ANOVA). Mean values of MDA at second time point between supplemented (2.37 ± 0.055, P \ 0.02) and control group (1.75 ± 0.067, P \ 0.001) and third time point (2.54 ± 0.055 and 2.17 ± 0.089) were significantly different.
Mda
Tac
Malondialdehyde level raised significantly within each group during the first 6 months, followed by a significant decline in
The TAC significantly increased during this study within both groups (P \ 0.001). Regarding between groups, it
intake between the two groups. The independent t test did not show any statistically significant differences, except for vitamin C in second time point assessment (P = 0.029).
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25.88 ± 0.17 vs. 25.86 ± 0.18 and in 12th month 26.8 ± 0.17 vs. 26.59 ± 0.18) were significant.
27.5 Supplemented
Control
MMSE (Score)
27 26.5 26
Discussion
25.5 25 24.5 24 0
6
12
Time points (Months)
Fig. 2 Mean and SE of MMSE score in the two studied groups at baseline and during the follow-up period, Asterisk significantly different from 0 to third time point (P \ 0.001) Repeated measures ANOVA), Double Asterisk significantly different from 0 to second time point (P \ 0.001) Repeated measures ANOVA)
reached to a significantly higher level in antioxidant received subjects compared with controls only at twelfth month of supplementation (P \ 0.001). GSH In GSH, within supplemented group using repeated contrasts showed significant differences between second and third time points (P = 0.02), while in control group, no significant differences were observed at all time points. The red blood cells glutathione was also significantly higher in supplemented group only at twelfth month of intervention compared with the control group (P \ 0.01). 8-OHdG The test showed that the difference of within group for 8-OHdG at second and third time point measurements compared with zero time was significant (P \ 0.001), but it did not show any significant differences between the two groups throughout the study (P \ 0.4). The results for the above biomarkers have been shown in Table 3. Mini-Mental State Examination (MMSE) score After adjusting for potential covariates including age, sex, BMI, blood pressure, educational levels, and dietary antioxidants intake, repeated measures ANOVA of the MMSE scores showed a significant (P \ 0.001) increasing trend within both supplemented and control groups throughout this study, but no significant differences between these groups were found (P = 0.88; Fig. 2). None of the mean values of MMSE score in 6th and 12th month of intervention between supplemented and control groups (In sixth month: supplemented vs. control
During 1-year, double-blind, randomized, controlled clinical trial, in spite of improvements in MDA, TAC, and GSH levels, no beneficial effect of antioxidants on cognition was observed at MMSE cognitive assessment. These markers are some of the most widely used tests of oxidative stress status and at least provide some indications of the bioavailability of administrated antioxidants, although changes in serum level of these markers could not be considered as a solid indication of redox status, especially at the exact target points of brain where antioxidants are expected to exert their possible cognitive effects. These results are in accordance with some parts of the previous studies, but disagree with others. Even detrimental consequences for antioxidants administration in MCI have been reported [56]. The real causes of these discrepancies among various experiments are unknown, but a long list of involving factors could be suspected. Duration of intervention, types and doses of antioxidants used, the efficacy of cognitive assessment tools, and the influence of confounding variables are some of the factors that alone, or in combination with each other, could potentially affect the outcomes. The quality of studies, especially the number of involving subjects, should also be added to the above list. Morris et al. [27] obtained positive results regarding the effect of vitamins E and C in Alzheimer disease. In their study, confounding factors may have adversely influenced and the results had been ignored. Alzheimer’s disease has been reported to be associated with educational attainment [28, 29], body mass index, and lifestyle factors such as physical activity [30, 31], older age, and sex [29, 32]. In our trial, all confounding variables were assessed and showed not to be statistically different in supplemented and control groups. Duration of intervention may be a major difference between our and some of the similar studies and may be one of the causes of difference in responsiveness to antioxidants supplementation. Grodstein et al. [18], who obtained the positive results, argued that the prolongation of antioxidant administration would increase the likelihood of appearance of their cognitive beneficial effect. Nevertheless, in some studies, even in spite of relatively long intervention period of 10 years [33], 3 years [22], 4 years [34] 5 years [35], no positive results were found. Therefore, supplementation duration alone may not always be a significant factor in effectiveness of antioxidants and must be noted along with other influencing factors. It is not clear
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that the prolongation of antioxidant supplementation beyond that of our study may result in any positive effect on cognition. In our trial, we selected 1-year period of antioxidant supplementation, and from our view point, it was an average duration considering the previous similar studies. Dosage of antioxidants can also be another important factor that seems to be remarkably effective in outcomes of this and similar studies. The relationship between antioxidants doses and cognitive performance in different studies has not been straightforward. These includes the negative result with 400 mg vitamin E alone [33], 1,400 mg vitamin E ? 10 mg donepezil [22] or 540 mg/d of vitamin E ? 500 mg/d of vitamin C ? 900 mg/d of a-lipoic acid ? 400 mg of coenzyme Q [36], and those including positive result with 270 mg vitamin E, 500 mg vitamin C [27] or 1,400 mg Vitamin E ? 10 mg Selegiline [21]. In regard to the above studies, some trials in spite of being categorized among high-dose users of antioxidants were not able to obtain positive result while some studies have been revealed negative results by using higher doses. Therefore, dosage of antioxidants cannot be the solely determinant of the outcome and must be noticed along with other effective variables. Higher dose of vitamin E ([ or = 270 mg/d) may increase mortality [37, 38] or increase the risk of prostate cancer [39]. High doses of vitamin C supplementation have been reported to be associated with a higher risk of agerelated cataract [40] or hyperoxaluric nephropathy and progressive renal failure [41]. In our study, supplemented group consumed 300 mg of vitamin E and 400 mg vitamin C per day. Vitamin E is considered relatively safe compared to other fat-soluble vitamins. Using a combination of vitamins E and C with a medium dose did not produce any unexpected side effects on subjects. Type of antioxidants used is another important factor that may influence the results. The selection of proper type(s) of antioxidant(s) for a study such as ours has always been a challenge. There is a long and increasing list of antioxidants, which seems proper candidate for studying MCI–antioxidant relation. Most studies that used just a-tocopherol in preventing Alzheimer’s disease and managing MCI indicated negative results [20, 22]. Therefore, in a newest study, authors recommended that future trials assessing vitamin E treatment in Alzheimer’s disease should not be restricted to alpha-tocopherol [42]. Betacarotene is also another antioxidant, which has been used by several studies in relation to MCI and Alzheimer’s disease [43–45]. Beta-carotene, due to its deleterious effect on smokers [46] and because of using vitamin E as a major lipophilic antioxidant similar to beta-carotene, was not used in our
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study. Due to the synergic effect of vitamin E ? C [35] and the fact that supplementation with vitamin E ? C has raised a-tocopherol level in CSF (45 %) than plasma (35 %) [47], these factors would have great importance to select as antioxidants for supplementation. It seems that other possible explanations exist for the ineffectiveness of antioxidants used on cognitive performance in our trial. For instance, aging is known to contribute significant vitamin E deficiencies, especially among elderly with MCI [6, 45, 47] also, and previous surveys indicated that oxidative stress is increasing in both aging [48, 49] and MCI [8, 50–53], thus depletion of vitamin reservoir in aging [54] and being exposed to oxidative stress may postpone the effect of antioxidants on their cognitive performance. Additionally, reduced absorption of several substances in aging [55] should also be noted. Strengths and weaknesses There are several limitations in our study. The most prominent of them was using the MMSE questionnaire as the sole tool for cognitive assessment, which may have reduced the chance of detecting small changes in cognitive abilities. In addition, another important point regarding using MMSE test has been shown in Fig. 2. As indicated in this figure, within subject variable of MMSE score in supplemented and control group was significant. It seems that participants have become familiar with the questions of this test following their first evaluation. This familiarity may affect the discriminatory efficacy of this test in subsequent evaluations and may act as a confounding factor. As one of the strengths of our study, cognitive evaluation was performed at three time points during the study. Other strengths of the current study could be mentioned as large enough sample size, measurement of oxidative stress biomarkers throughout the study [56], administration of two different antioxidant vitamins, relatively long study period, and controlling several confounding variables. In most previous studies, many of these factors have been ignored.
Conclusions Epidemiological or clinical studies have not provided the ultimate answer to whether antioxidants can truly improve the cognitive performance or not. This may be attributable to several reasons listed in the text. In our study, despite some evidences of alterations in oxidative stress markers toward a reduced status, there was not any remarkable effect on cognitive function, although several confounding factors were controlled. A large number of kinetic and/or dynamic factors could be suspected for this
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unresponsiveness. Further studies especially with more efficient cognitive evaluation tools as well as more diverse antioxidant types may be needed to clarify the possible antioxidant–oxidative stress cognition interactions. Acknowledgments This study was supported by Institute of Nutritional Sciences, University of Vienna and the Vice-chancellor for Research, Tehran University of Medical Sciences (TUMS), Iran, by a Grant (No. 11126). Conflict of interest: of interest.
The authors declare that there are no conflicts
References 1. WHO (2012) Dementia: A public health priority: http://whqlib doc.who.int/publications/2012/9789241564458_eng.pdf 2. Mecocci P, Polidorib MC (2012) Antioxidant clinical trials in mild cognitive impairment and Alzheimer’s disease. Biochim Biophys Acta 5:631–638. doi:10.1016/j.bbadis.2011.10.006 3. Nelson AP, O’Connor MG (2008) Mild cognitive impairment: a neuropsychological perspective. CNS Spectr 13(1):56–64 4. Padurariu M, Ciobica A, Hritcu L, Stoica B, Bild W, Stefanescu C (2010) Changes of some oxidative stress markers in the serum of patients with mild cognitive impairment and Alzheimer’s disease. Neurosci Lett 18(469(1)):6–10 5. Smith GE, Petersen RC, Parisi JE, Ivnik RJ (1996) Definition, course, and outcome of mild cognitive impairment. Aging Neuropsychol Cogn 3(2):141–147. doi:10.1080/13825589608256619 6. Baldeiras I, Santana I, Proenc¸a MT, Garrucho MH, Pascoal R, Rodrigues A, Duro D, Oliveira CR (2008) Peripheral oxidative damage in mild cognitive impairment and mild Alzheimer’s disease. J Alzheimers Dis 15(1):117–128 7. Lovell MA, Markesbery WR (2008) Oxidatively modified RNA in mild cognitive impairment. Neurobiol Dis 29(2):169–175. doi:10.1016/j.nbd.2007.07.030 8. Torres LL, Quaglio NB, de Souza GT et al (2011) Oxidative stress biomarkers in mild cognitive impairment and Alzheimer’s disease. J Alzheimers Dis 26(1):59–68 9. Shao C, Xiong S, Li GM, Gu L, Mao G, Markesbery WR, Lovell MA (2008) Altered 8-oxoguanine glycosylase in mild cognitive impairment and late-stage Alzheimer’s disease brain. Free Radic Biol Med 45(6):813–819. doi:10.1016/j.freeradbiomed.2008.06. 003 10. Baldeiras I, Santana I, Proenc¸a MT, Garrucho MH, Pascoal R, Rodrigues A, Duro D, Oliveira CR (2010) Oxidative damage and progression to Alzheimer’s disease in patients with mild cognitive impairment. J Alzheimers Dis 21(4):1165–1177 11. Guidi I, Galimberti D, Lonati S et al (2006) Oxidative imbalance in patients with mild cognitive impairment and Alzheimer’s disease. Neurobiol Aging 27(2):262–269. doi:10.1016/j.neuro biolaging.2005.01.001 12. Lo´pez N, Tormo C, De Blas I, Llinares I, Alom J (2013) Oxidative stress in Alzheimer’s disease and mild cognitive impairment with high sensitivity and specificity. J Alzheimers Dis 33(3):823–829. doi:10.3233/JAD-2012-121528 13. Cervellati C, Cremonini E, Bosi C, Magon S, Zurlo A, Bergamini CM, Zuliani G (2012) Systemic Oxidative Stress in Older Patients with Mild Cognitive Impairment or Late Onset Alzheimer’s Disease. Curr Alzheimer Res 14. Stocker R (2007) Vitamin E. Novartis Found Symp 282:77–87. doi:10.1002/9780470319444.ch6
15. Masaki KH, Losonczy KG, Izrnirlian G (2000) Association of vitamin E and C supplement use with cognitive function and dementia in elderly men. Neuroiem 54:1265–1272 16. Morris MC, Evans DA, Bienias JL, Tangney CC, Wilson RS (2002) Vitamin E and cognitive decline in older persons. Arch Newrol 59(7):1125–1132. doi:10.1001/archneur.59.7.1125 17. Engelhart MJ, Geerlings MI, Ruitenberg A (2002) Dietary intake of antioxidants and risk of Alzheimer disease. JAMA 287(24):3223–3229. doi:10.1001/jama.287.24.3223 18. Grodstein F, Chen J, Willett WC (2003) High-dose antioxidant supplements and cognitive function in community-dwelling elderly women. Am J Clin Nutr 77(4):975–984 19. Zandi PP, Anthony JC, Khachaturian AS et al (2004) Reduced risk of Alzheimer disease in users of antioxidant vitamin supplements: the cache county study. Arch Neurol 61(1):82–88. doi:10.1001/archneur.61.1.82 20. Isaac MG, Quinn R, Tabet N (2008) Vitamin E for Alzheimer’s disease and mild cognitive impairment. Cochrane database Syst Rev 16 (3):CD002854. doi: 10.1002/14651858.CD002854.pub2 21. Sano M, Ernesto C, Thomas RG et al (1997) 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(17):1216–1222. doi:10.1056/NEJM199704243361704 22. Petersen RC, Thomas RG, Grundman M et al (2005) Vitamin E and donepezil for the treatment of mild cognitive impairment. N Engl J Med 352(23):2379–2388. doi:10.1056/NEJMoa050151 23. Seyedian M, Falamh M, Nourouzian M, Nejat S, Delavar A, Ghasemzadeh HA (2008) Validity of the Farsi version of MiniMental State Examination. J Med Counc I.R.I. 25(4):408–414 (Abstract in English) 24. Yu LW, Latriano L, Duncan S, Hartwick RA, Witz G (1986) High-performance liquid chromatography analysis of the thiobarbituric acid adducts of malondialdehyde and trans, trans-muconaldehyde. Anal Biochem 156(2):326–333 25. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A (1993) A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci (lond) 84(4):407–412 26. Beutler E, Duron O, Kelly BM (1963) Improved method for the determination of blood glutathione. J Lab Clin Med 61:882–888 27. Morris MC, Beckett LA, Scherr PA, Hebert LE, Bennett DA, Field TS, Evans DA (1998) Vitamin E and vitamin C supplement use and risk of incident Alzheimer disease. Alzheimer Dis Assoc Disord 12(3):121–126. doi:10.1097/00002093-19980900000001 28. Sattler C, Toro P, Scho¨nknecht P, Schro¨der J (2012) Cognitive activity, education and socioeconomic status as preventive factors for mild cognitive impairment and Alzheimer’s disease. Psychiatry Res 196(1):90–95. doi:10.1016/j.psychres.2011.11.012 29. Letenneur L, Gilleron V, Commenges D, Helmer C, Orgogozo J, Dartigues J (1999) Are sex and educational level independent predictors of dementia and Alzheimer’s disease? Incidence data from the PAQUID project. J Neurol Neurosurg Psychiatry 66(2):177–183. doi:10.1136/jnnp.66.2.177 30. Scarmeas N, Luchsinger JA, Schup FN, Brickman AM, Cosentino S, Tang MX, Stern Y (2009) Physical activity, diet, and risk of Alzheimer disease. JAMA 302(6):627–637. doi:10.1001/jama 2009.1144 31. Buchman AS, Wilson RS, Bienias JL, Shah RC, Evans DA, Bennett DA (2005) Change in body mass index and risk of incident Alzheimer disease. Neurology 65(6):892–897. doi:10. 1212/01.wnl.0000176061.33817.90 32. Kukull WA, Higdon R, Bowen JD et al (2002) Dementia and Alzheimer disease incidence: a prospective cohort study. Arch Neurol 59(11):1737–1746. doi:10.1001/archneur.59.11.1737
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Eur J Nutr 33. Kang JH, Cook N, Manson J, Buring JE, Grodstein F (2006) A randomized trial of vitamin E supplementation and cognitive function in women. Arch Intern Med 166(22):2462–2468. doi:10. 1001/archinte.166.22.2462 34. Luchsinger JA, Tang MX, Shea S, Mayeux R (2003) Antioxidant vitamin intake and risk of Alzheimer disease. Arch Neurol 60(2):203–208. doi:10.1001/archneur.60.2.203 35. Maxwell CJ, Hicks MS, Hogan DB, Basran J (2005) Supplemental use of antioxidant vitamins and subsequent risk of cognitive decline and dementia. Dement Geriatr Cogn Disord 20(1):45–51. doi:10.1159/000085074 36. Galasko DR, Peskind E, Clark CM et al (2012) Antioxidants for Alzheimer’disease randomized clinical trial with cerebrospinal fluid biomarker measures. Arch Neurol 69(7):836–841. doi:10. 1001/archneurol.2012.85 37. Miller ER 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E (2005) Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 142(1):37–46 38. Lock M, Loblaw A (2005) Vitamin E might increase risk of death. Can Fam Physician 51(6):829–831 39. Klein EA, Thompson IM, Tangen CM et al (2011) Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 306(14):1549–1556. doi:10. 1001/jama 2011.1437 40. Rautiainen S, Lindblad BE, Morgenstern R, Wolk A (2010) Vitamin C supplements and the risk of age-related cataract: a population-based prospective cohort study in women. Am J Clin Nutr 91(2):487–493. doi:10.3945/ajcn.2009.28528 41. Rathi S, Kern W, Lau K (2007) Vitamin C-induced hyperoxaluria causing reversible tubulointerstitial nephritis and chronic renal failure: a case report. J Med Case Rep 1:155. doi:10.1186/17521947-1-155 42. Farina N, Isaac MG, Clark AR, Rusted J, Tabet N (2012) Vitamin E for Alzheimer’s dementia and mild cognitive impairment. Cochrane Database of Systematic Reviews 11; CD002854. doi: 10.1002/14651858.CD002854.pub3 43. Stuerenburg HJ, Ganzer S, Mu¨ller-Thomsen T (2005) Plasma beta carotene in Alzheimer’s disease. Association with cerebrospinal fluid beta-amyloid 1-40, (Abeta40), beta-amyloid 1-42 (Abeta42) and total Tau. Neuro Endocrinol Lett 26(6):696–698 44. de Oliveira BF, Veloso CA, Nogueira-Machado JA, de Moraes EN, Santos RR, Cintra MT, Chaves MM (2012) Ascorbic acid, alpha-tocopherol, and beta-carotene reduce oxidative stress and proinflammatory cytokines in mononuclear cells of Alzheimer’s
123
45.
46.
47.
48. 49.
50.
51.
52.
53.
54.
55.
56.
disease patients. Nutr Neurosci. doi:10.1179/1476830512Y. 0000000019 Rinaldi P, Polidori MC, Metastasio A et al (2003) Plasma antioxidants are similarly depleted in mild cognitive impairment and in Alzheimer’s disease. Neurobiol Aging 24(7):915–919. doi:10. 1016/S0197-4580(03)00031-9 Arora A, Willhite CA, Liebler DC (2001) Interactions of betacarotene and cigarette smoke in human bronchial epithelial cells. Carcinogenesis 22(8):1173–1178. doi:10.1093/carcin/22.8.1173 Kontush A, Mann U, Arlt S, Ujeyl A, Lu¨hrs C, Mu¨ller-Thomsen T, Beisiegel U (2001) Influence of vitamin E and C supplementation on lipoprotein oxidation in patients with Alzheimer’s disease. Free Radic Biol Med 31(3):345–354. doi:10.1016/S08915849(01)00595-0 Giblin FJ (2000) Glutathione: a vital lens antioxidant. J Ocul Pharmacol Ther 16(2):121–135 Spiteller G (2001) Lipid peroxidation in aging and age-dependent diseases. Exp Gerontol 36(9):1425–1457. doi:10.1016/S05315565(01)00131-0 Pratico` D, Clark CM, Liun F, Rokach J, Lee VY, Trojanowski JQ (2002) Increase of brain oxidative stress in mild cognitive impairment: a possible predictor of Alzheimer disease. Arch Neurol 59(6):972–976. doi:10.1001/archneur.59.6.972 Montine KS, Quinn JF, Zhang J, Fessel JP, Roberts LJ, Morrow JD, Montin TJ (2004) Isoprostanes and related products of lipid peroxidation in neurodegenerative diseases. Chem Phys Lipids 128:117–124. doi:10.1016/j.chemphyslip.2003.10.010 Keller JN, Schmitt FA, Scheff SW, Ding Q, Chen Q, Butterfield DA, Markesbery WR (2005) Evidence of increased oxidative damage in subjects with mild cognitive impairment. Neurology 64(7):1152–1156. doi:10.1212/01.WNL.0000156156.13641.BA Markesbery WR, Lovell MA (2007) Damage to lipids, proteins, DNA, and RNA in mild cognitive impairment. Arch Neurol 64(7):954–956. doi:10.1001/archneur.64.7.954 Rath M (2003) External and inherited cardiovascular risks: in: Rath M, 4th eds. Why animals don’t get heart attacks, published by Dr. Rath Health Foundation Husebye E, Engedal K (1992) The patterns of motility are maintained in the human small intestine throughout the process of aging. Scand J Gastroenterol 27(5):397–404. doi:10.3109/ 00365529209000095 Lloret A, Badı´a MC, Mora NJ, Pallardo´ FV, Alonso MD, Vin˜a J (2009) Vitamin E paradox in Alzheimer’s disease: it does not prevent loss of cognition and may even be detrimental. J Alzheimers Dis 17(1):143–149
ORIGINAL CONTRIBUTION
The effect of antioxidant vitamins E and C on cognitive performance of the elderly with mild cognitive impairment in Isfahan, Iran: a double-blind, randomized, placebo-controlled trial A. M. Alavi Naeini • I. Elmadfa • A. Djazayery M. Barekatain • M. R. Aghaye Ghazvini • M. Djalali • A. Feizi
•
Received: 15 June 2013 / Accepted: 18 November 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose This study was carried out to investigate the effect of vitamins E and C on cognitive performance among the elderly in Iran. Methods About 256 elderly with mild cognitive impairment, aged 60–75 years, received 300 mg of vitamin E plus 400 mg of vitamin C or placebo daily just for 1 year. Background Demographic characteristics, anthropometric variables food consumption, cognitive function by Mini-Mental State Examination (MMSE), and some of the oxidative stress biomarkers were examined. Results Antioxidant supplementation reduced malondialdehyde level (P \ 0.001) and raised total antioxidant capacity (P \ 0.001) and glutathione (P \ 0.01). The A. M. Alavi Naeini A. Djazayery M. Djalali School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, Iran I. Elmadfa Institute of Nutritional Sciences, University of Vienna, Vienna, Austria I. Elmadfa (&) International Union of Nutritional Sciences (IUNS), Althanstrasse 14, 1090 Vienna, Austria e-mail: [email protected] M. Barekatain Department of Psychiatry and Behavioral Sciences, Isfahan University of Medical Sciences, Isfahan, Iran M. R. Aghaye Ghazvini Isfahan Center of Health Research, Nationally Institute of Health Research, Tehran University of Medical Science, Tehran, Iran A. Feizi Department of Biostatistics and Epidemiology, Isfahan University of Medical Sciences, Isfahan, Iran
serum 8-hydroxydeoxyguanosine remained unchanged (P \ 0.4). After adjusting for the covariates effects, MMSE scores following 6- (25.88 ± 0.17) and 12-month antioxidant supplementation (26.8 ± 0.17) did not differ from control group (25.86 ± 0.18 and 26.59 ± 0.18, respectively). Conclusion Despite significant improvement in most of the oxidative stress biomarkers, antioxidants’ supplementation was not observed to enhance cognitive performance. A large number of kinetic and/or dynamic factors could be suspected. Keywords Supplementation The elderly Mild cognitive impairment Vitamin E Vitamin C
Introduction Dementia is defined as a progressive loss of brain cognitive function that mainly affects older people. It can be occurred by a number of etiologies, which lead to a decline in memory, judgment, decision making, learning, executive functioning, and other mental activities. The symptoms would cause clinically significant distress or impairment in social, occupational, or other important areas of functioning [1]. Alzheimer’s disease, for example, is the most known cause of dementia, characterized by severe cognitive and functional disabilities [2]. As a slowly progressive process, cognitive decline in its early stages may not impair important area of functioning. Mild cognitive impairment (MCI) is another term, which has been defined as a transitional stage between normal cognitive function and dementia. People with MCI have minor problems with memory, attention, speech, decision making, visuospatial, and psychomotor functions, which exceed those expected
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for their age and educational level [3]. However, there is no sign of major impairment in social, occupational, or personal functioning. The patients suffering from MCI have a significantly higher rate, about tenfold, of progression to Alzheimer’s disease compared to cognitively normal elderly people [4, 5]. Despite considerable investigations, the exact factors associated the induction of MCI or its more rapid progression to Alzheimer’s disease has not been well defined. Extensive evidence indicates that oxidative stress plays an important role in the pathogenesis of cognitive disorders. Oxidative stress has been defined as a state where highly reactive free radicals activity exceeds the antioxidant systems defense. This can lead to chemical oxidative modification of macromolecules and change the structure and function of cellular key components [4, 6, 7]. Increased level of oxidative stress markers including malondialdehyde (MDA) [4, 8] and (8-hydroxydeoxyguanosine) 8-OHdG [9] and decreased levels of glutathione (GSH) [10], total antioxidant capacity (TAC) [11], and antioxidant enzymes [4] have been reported in MCI patients. Cerebral tissue is particularly susceptible to reactive oxygen spices (ROS) damage due to its high oxygen requirements for metabolism, low content of antioxidants, and a high content of polyunsaturated fatty acids [4, 6, 7]. These oxidative events have been implicated both in MCI [12] and its progression to Alzheimer’s disease [13]. Regarding these observations, antioxidant vitamins, including vitamins E, the most potent lipophilic chainbreaking antioxidant [14], and vitamin C, a potent watersoluble antioxidant, are expected to reduce neuronal damage and improve cognitive performance of these patients; however, previous studies have yielded conflicting results in this regard. While some clinical and epidemiological studies pointed to the slowing of cognitive decline with the use of vitamins C and/or E [15–19], some others failed to find any protective effect for these antioxidants [20–22]. The aim of the present study was to examine the effects of antioxidant vitamins E and C supplements on cognitive performance in a group of elderly males and females in Iran.
disease, Alcohol intake, smoking, and routine consumption of neurological or antioxidants drugs. An expert psychologist evaluated the subjects to find those suffering from MCI on the basis of attaining 21–26 scores in Mini-Mental State Examination (MMSE). The Iranian version of MMSE had been validated and found to be applicable for the local elderly population [23]. Of the 761 volunteers, 296 were found to have MCI and selected for the next part of the study. From these, 40 did not continue with the study due to the problems tolerating the supplementations (14 subjects), 1 subject died, and 25 subjects abstained to continue participation due to personal reasons. Therefore, 256 subjects completed the study. As shown in Fig. 1, finally selected subjects were randomly assigned into two groups, namely intervention and control. Selection and grouping were performed using stratified method followed by simple randomization method. The present study protocol was conducted in accordance with the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Ethics Committee at Ministry of Health and Medical Education, Iran. To participate in this study, all the subjects or their families signed the consent forms.
Subjects and methods
Data analysis
Subjects and study design
The results are presented as mean ± standard error of mean (SEM) for quantitative variables and number (percent) for qualitative variables. Repeated measures analysis of variance (ANOVA) was used for analyzing the data as the main statistical method. Mauchly’s sphericity test was conducted to assess sphericity as a perquisite assumption.
This study was designed as a double-blind, randomized, controlled clinical trial. From retirees clubs, 761 elderly volunteers with ages between 60 and 75 years were recruited. Exclusion criteria included obvious disabling
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Procedures One group consumed 300 mg of vitamin E (Dl-alphatocopherol acetate) plus 400 mg vitamin C (ascorbic acid) per day, and the second group consumed placebo with the identical condition over the 1-year intervention period. Fasting venous blood from each subject was obtained at three time points, namely on days 0, 180, and 360. Serum MDA was measured according to the method described by Yu et al. [24]. TAC was assessed based on Miller and RiceEvans [25]. Red blood cells GSH was determined quantitatively using PerkinElmer spectrophotometer according to the method of Beutler et al. [26]. 8-OHdG was measured using a competitive enzyme-linked immunosorbent assay (ELISA) kit from Immundiagnostik Company, Germany. Cognitive performance was evaluated by MMSE at three time points similar to blood sampling. Three-day dietary record forms were also completed for each participant by a trained nutritionist every 2 months throughout the study.
Eur J Nutr Fig. 1 Stratified and simple randomization
Table 1 Descriptive characteristics of the subjects at baseline Characteristic
Supplemented (n = 127)
Control (n = 129)
P value*
Age (year) Weight (kg)
66.5 ± 0.39 71.0 ± 0.94
66.3 ± 0.38 69.1 ± 0.94
0.755 0.139 0.703
Height (cm)
162.5 ± 0.78
162 ± 0.86
BMI (kg/m2)
26.9 ± 0.35
26.3 ± 0.35
0.233
Systolic blood pressure
141.2 ± 1.67
137.4 ± 1.7
0.119
Diastolic blood pressure
80.2 ± 1.2
77.9 ± 1.2
0.164
Male n (%)
63 (49.6)
57 (44.2)
0.229
Female n (%)
64 (50.4)
72 (55.8)
25 (19.8)
16 (12.5)
Sex
Results General characteristic of the subjects
Educational level Primary n (%)
When the assumption was not satisfied, Huynh–Feldt correction was used. Within-group comparisons at each follow-up time point were made using repeated contrasts. Between-group comparisons were conducted using two independent samples t test. Chi square test for difference between proportions was used. Analysis of the data was done by SPSS software version 19. Analysis of consumed foods was done by Food Processor II software. For all hypotheses, a significance level of P \ 0.05 was considered statistically significant.
Secondary n (%)
17 (13.5)
10 (7.8)
Diploma n (%)
48 (38.1)
55 (43.0)
University degree n (%)
36 (28.6)
47 (36.7)
* Student’s t test or chi–square test
0.127
General characteristics of the studied subjects for both groups are summarized in Table 1. As can be seen, no significant differences were found between supplemented and control groups (Student’s t test or Chi square tests). Data from 3-day dietary record for intakes of vitamin C, vitamin E, carotene, selenium, and zinc were analyzed (Table 2) to compare the dietary antioxidant
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Eur J Nutr Table 2 Means and standard error of dietary intake antioxidants based on 3-day dietary record every 2 month during 1 year for supplemented and control groups Variable groups Vitamin E (mg)
Vitamin C (mg)
Carotene (mg)
Selenium (lg)
Zinc (mg)
Supplemented
First time
Second time
Third time
Fourth time
Fifth time
Sixth time
12.0 ± 0.9
12.2 ± 1.0
10.7 ± 0.4
11.9 ± 0.9
10.0 ± 0.5
10.4 ± 0.4
Control
11.6 ± 0.8
10.0 ± 0.4
10.4 ± 0.4
10.2 ± 0.4
10.6 ± 0.6
10.2 ± 0.4
P value
0.357
0.134
0.672
0.625
0.433
0.134
Supplemented
90.7 ± 6.4
97.0 ± 6.5
92.6 ± 5.9
118.4 ± 20.1
103.5 ± 6.2
107 ± 5.6
Control
98.6 ± 5.8
110.7 ± 6.4
99.9 ± 5.9
108.4 ± 6.2
117.6 ± 6.8
109 ± 5.4
P value
0.729
0.029*
0.376
0.066
0.126
0.779
Supplemented Control
759 ± 63.6 687 ± 54.2
668 ± 62.1 653 ± 59.1
575 ± 47.6 647 ± 52.5
593 ± 46.5 589 ± 48.6
594 ± 55.0 566 ± 46.5
502 ± 43.4 540 ± 42.5
P value
0.388
0.866
0.306
0.960
0.698
0.525
Supplemented
69.3 ± 4.1
75.6 ± 5.0
78.0 ± 6.0
84.7 ± 6.1
74.6 ± 6.0
70.2 ± 4.1
Control
67.7 ± 4.2
66.9 ± 4.3
72.0 ± 4.8
70.0 ± 4.7
66.3 ± 4.9
74.1 ± 4.9
P value
0.787
0.188
0.415
0.550
0.281
0.552
Supplemented
9.2 ± 0.3
8.9 ± 0.4
8.7 ± 0.3
9.6 ± 0.7
8.4 ± 0.3
8.6 ± 0.3
Control
9.1 ± 0.4
8.6 ± 0.3
9.6 ± 0.7
8.7 ± 0.3
8.4 ± 0.3
8.9 ± 0.3
* Independent t test P \ 0.029
Table 3 Results of within- and between-group comparisons for the MDA, TAC, GSH, and 8-OHdG based on repeated measures ANOVA Variable groups MDA
TAC
GSH
8-OHdG
Period 0
Period 180
Period 360
Time
Treatment
Time 9 treatment
Supplemented
1.54 ± 0.054
2.37 ± 0.055
1.75 ± 0.067
F = 153.7
F = 9.49
F = 2.44
Control
1.56 ± 0.052
2.54 ± 0.055
2.17 ± 0.089
P \ 0.001**
P = 0.002**
P = 0.12
t value
t = 0.27
t = 2.3
t = 3.8
P value
P = 0.78
P = 0.02
P \ 0.001
Supplemented
1.08 ± 0.022
1.47 ± 0.028
1.78 ± 0.048
F = 241.0
F = 16.77
F = 16.38
Control
1.06 ± 0.019
1.43 ± 0.027
1.49 ± 0.031
P \ 0.001**
P \ 0.001**
P \ 0.001**
t value P value
t = -652 P \ 0.51
t = -1.02 P \ 0.31
t = -5.17 P \ 0.001*
Supplemented
61.7 ± 2.84
58.9 ± 1.25
64.5 ± 1.38
F = 1.683
F = 0.236
F = 5.580
Control
59.7 ± 2.27
60.9 ± 1.31
59.7 ± 1.25
P = 0.18
P = 0.63
P = 0.02**
t value
t = -0.529
t = 1.23
t = -2.52
P value
P \ 0.59
P \ 0.22
P \ 0.001*
Supplemented
0.129 ± 0.007
0.097 ± 0.003
0.082 ± 0.003
F = 31.53
F = 0.710
F = 0.976
Control
0.134 ± 0.010
0.097 ± 0.003
0.092 ± 0.007
P \ 0.001**
P = 0.401
P = 0.350
t value
t = -0.359
T = 0.019
t = -1.329
P value
P \ 0.72
P \ 0.98
P \ 0.18
** Repeated measure ANOVA, * Student’s t test
Oxidative stress biomarkers
second half-year of the study (P \ 0.001, repeated measures ANOVA). Mean values of MDA at second time point between supplemented (2.37 ± 0.055, P \ 0.02) and control group (1.75 ± 0.067, P \ 0.001) and third time point (2.54 ± 0.055 and 2.17 ± 0.089) were significantly different.
Mda
Tac
Malondialdehyde level raised significantly within each group during the first 6 months, followed by a significant decline in
The TAC significantly increased during this study within both groups (P \ 0.001). Regarding between groups, it
intake between the two groups. The independent t test did not show any statistically significant differences, except for vitamin C in second time point assessment (P = 0.029).
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25.88 ± 0.17 vs. 25.86 ± 0.18 and in 12th month 26.8 ± 0.17 vs. 26.59 ± 0.18) were significant.
27.5 Supplemented
Control
MMSE (Score)
27 26.5 26
Discussion
25.5 25 24.5 24 0
6
12
Time points (Months)
Fig. 2 Mean and SE of MMSE score in the two studied groups at baseline and during the follow-up period, Asterisk significantly different from 0 to third time point (P \ 0.001) Repeated measures ANOVA), Double Asterisk significantly different from 0 to second time point (P \ 0.001) Repeated measures ANOVA)
reached to a significantly higher level in antioxidant received subjects compared with controls only at twelfth month of supplementation (P \ 0.001). GSH In GSH, within supplemented group using repeated contrasts showed significant differences between second and third time points (P = 0.02), while in control group, no significant differences were observed at all time points. The red blood cells glutathione was also significantly higher in supplemented group only at twelfth month of intervention compared with the control group (P \ 0.01). 8-OHdG The test showed that the difference of within group for 8-OHdG at second and third time point measurements compared with zero time was significant (P \ 0.001), but it did not show any significant differences between the two groups throughout the study (P \ 0.4). The results for the above biomarkers have been shown in Table 3. Mini-Mental State Examination (MMSE) score After adjusting for potential covariates including age, sex, BMI, blood pressure, educational levels, and dietary antioxidants intake, repeated measures ANOVA of the MMSE scores showed a significant (P \ 0.001) increasing trend within both supplemented and control groups throughout this study, but no significant differences between these groups were found (P = 0.88; Fig. 2). None of the mean values of MMSE score in 6th and 12th month of intervention between supplemented and control groups (In sixth month: supplemented vs. control
During 1-year, double-blind, randomized, controlled clinical trial, in spite of improvements in MDA, TAC, and GSH levels, no beneficial effect of antioxidants on cognition was observed at MMSE cognitive assessment. These markers are some of the most widely used tests of oxidative stress status and at least provide some indications of the bioavailability of administrated antioxidants, although changes in serum level of these markers could not be considered as a solid indication of redox status, especially at the exact target points of brain where antioxidants are expected to exert their possible cognitive effects. These results are in accordance with some parts of the previous studies, but disagree with others. Even detrimental consequences for antioxidants administration in MCI have been reported [56]. The real causes of these discrepancies among various experiments are unknown, but a long list of involving factors could be suspected. Duration of intervention, types and doses of antioxidants used, the efficacy of cognitive assessment tools, and the influence of confounding variables are some of the factors that alone, or in combination with each other, could potentially affect the outcomes. The quality of studies, especially the number of involving subjects, should also be added to the above list. Morris et al. [27] obtained positive results regarding the effect of vitamins E and C in Alzheimer disease. In their study, confounding factors may have adversely influenced and the results had been ignored. Alzheimer’s disease has been reported to be associated with educational attainment [28, 29], body mass index, and lifestyle factors such as physical activity [30, 31], older age, and sex [29, 32]. In our trial, all confounding variables were assessed and showed not to be statistically different in supplemented and control groups. Duration of intervention may be a major difference between our and some of the similar studies and may be one of the causes of difference in responsiveness to antioxidants supplementation. Grodstein et al. [18], who obtained the positive results, argued that the prolongation of antioxidant administration would increase the likelihood of appearance of their cognitive beneficial effect. Nevertheless, in some studies, even in spite of relatively long intervention period of 10 years [33], 3 years [22], 4 years [34] 5 years [35], no positive results were found. Therefore, supplementation duration alone may not always be a significant factor in effectiveness of antioxidants and must be noted along with other influencing factors. It is not clear
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that the prolongation of antioxidant supplementation beyond that of our study may result in any positive effect on cognition. In our trial, we selected 1-year period of antioxidant supplementation, and from our view point, it was an average duration considering the previous similar studies. Dosage of antioxidants can also be another important factor that seems to be remarkably effective in outcomes of this and similar studies. The relationship between antioxidants doses and cognitive performance in different studies has not been straightforward. These includes the negative result with 400 mg vitamin E alone [33], 1,400 mg vitamin E ? 10 mg donepezil [22] or 540 mg/d of vitamin E ? 500 mg/d of vitamin C ? 900 mg/d of a-lipoic acid ? 400 mg of coenzyme Q [36], and those including positive result with 270 mg vitamin E, 500 mg vitamin C [27] or 1,400 mg Vitamin E ? 10 mg Selegiline [21]. In regard to the above studies, some trials in spite of being categorized among high-dose users of antioxidants were not able to obtain positive result while some studies have been revealed negative results by using higher doses. Therefore, dosage of antioxidants cannot be the solely determinant of the outcome and must be noticed along with other effective variables. Higher dose of vitamin E ([ or = 270 mg/d) may increase mortality [37, 38] or increase the risk of prostate cancer [39]. High doses of vitamin C supplementation have been reported to be associated with a higher risk of agerelated cataract [40] or hyperoxaluric nephropathy and progressive renal failure [41]. In our study, supplemented group consumed 300 mg of vitamin E and 400 mg vitamin C per day. Vitamin E is considered relatively safe compared to other fat-soluble vitamins. Using a combination of vitamins E and C with a medium dose did not produce any unexpected side effects on subjects. Type of antioxidants used is another important factor that may influence the results. The selection of proper type(s) of antioxidant(s) for a study such as ours has always been a challenge. There is a long and increasing list of antioxidants, which seems proper candidate for studying MCI–antioxidant relation. Most studies that used just a-tocopherol in preventing Alzheimer’s disease and managing MCI indicated negative results [20, 22]. Therefore, in a newest study, authors recommended that future trials assessing vitamin E treatment in Alzheimer’s disease should not be restricted to alpha-tocopherol [42]. Betacarotene is also another antioxidant, which has been used by several studies in relation to MCI and Alzheimer’s disease [43–45]. Beta-carotene, due to its deleterious effect on smokers [46] and because of using vitamin E as a major lipophilic antioxidant similar to beta-carotene, was not used in our
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study. Due to the synergic effect of vitamin E ? C [35] and the fact that supplementation with vitamin E ? C has raised a-tocopherol level in CSF (45 %) than plasma (35 %) [47], these factors would have great importance to select as antioxidants for supplementation. It seems that other possible explanations exist for the ineffectiveness of antioxidants used on cognitive performance in our trial. For instance, aging is known to contribute significant vitamin E deficiencies, especially among elderly with MCI [6, 45, 47] also, and previous surveys indicated that oxidative stress is increasing in both aging [48, 49] and MCI [8, 50–53], thus depletion of vitamin reservoir in aging [54] and being exposed to oxidative stress may postpone the effect of antioxidants on their cognitive performance. Additionally, reduced absorption of several substances in aging [55] should also be noted. Strengths and weaknesses There are several limitations in our study. The most prominent of them was using the MMSE questionnaire as the sole tool for cognitive assessment, which may have reduced the chance of detecting small changes in cognitive abilities. In addition, another important point regarding using MMSE test has been shown in Fig. 2. As indicated in this figure, within subject variable of MMSE score in supplemented and control group was significant. It seems that participants have become familiar with the questions of this test following their first evaluation. This familiarity may affect the discriminatory efficacy of this test in subsequent evaluations and may act as a confounding factor. As one of the strengths of our study, cognitive evaluation was performed at three time points during the study. Other strengths of the current study could be mentioned as large enough sample size, measurement of oxidative stress biomarkers throughout the study [56], administration of two different antioxidant vitamins, relatively long study period, and controlling several confounding variables. In most previous studies, many of these factors have been ignored.
Conclusions Epidemiological or clinical studies have not provided the ultimate answer to whether antioxidants can truly improve the cognitive performance or not. This may be attributable to several reasons listed in the text. In our study, despite some evidences of alterations in oxidative stress markers toward a reduced status, there was not any remarkable effect on cognitive function, although several confounding factors were controlled. A large number of kinetic and/or dynamic factors could be suspected for this
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unresponsiveness. Further studies especially with more efficient cognitive evaluation tools as well as more diverse antioxidant types may be needed to clarify the possible antioxidant–oxidative stress cognition interactions. Acknowledgments This study was supported by Institute of Nutritional Sciences, University of Vienna and the Vice-chancellor for Research, Tehran University of Medical Sciences (TUMS), Iran, by a Grant (No. 11126). Conflict of interest: of interest.
The authors declare that there are no conflicts
References 1. WHO (2012) Dementia: A public health priority: http://whqlib doc.who.int/publications/2012/9789241564458_eng.pdf 2. Mecocci P, Polidorib MC (2012) Antioxidant clinical trials in mild cognitive impairment and Alzheimer’s disease. Biochim Biophys Acta 5:631–638. doi:10.1016/j.bbadis.2011.10.006 3. Nelson AP, O’Connor MG (2008) Mild cognitive impairment: a neuropsychological perspective. CNS Spectr 13(1):56–64 4. Padurariu M, Ciobica A, Hritcu L, Stoica B, Bild W, Stefanescu C (2010) Changes of some oxidative stress markers in the serum of patients with mild cognitive impairment and Alzheimer’s disease. Neurosci Lett 18(469(1)):6–10 5. Smith GE, Petersen RC, Parisi JE, Ivnik RJ (1996) Definition, course, and outcome of mild cognitive impairment. Aging Neuropsychol Cogn 3(2):141–147. doi:10.1080/13825589608256619 6. Baldeiras I, Santana I, Proenc¸a MT, Garrucho MH, Pascoal R, Rodrigues A, Duro D, Oliveira CR (2008) Peripheral oxidative damage in mild cognitive impairment and mild Alzheimer’s disease. J Alzheimers Dis 15(1):117–128 7. Lovell MA, Markesbery WR (2008) Oxidatively modified RNA in mild cognitive impairment. Neurobiol Dis 29(2):169–175. doi:10.1016/j.nbd.2007.07.030 8. Torres LL, Quaglio NB, de Souza GT et al (2011) Oxidative stress biomarkers in mild cognitive impairment and Alzheimer’s disease. J Alzheimers Dis 26(1):59–68 9. Shao C, Xiong S, Li GM, Gu L, Mao G, Markesbery WR, Lovell MA (2008) Altered 8-oxoguanine glycosylase in mild cognitive impairment and late-stage Alzheimer’s disease brain. Free Radic Biol Med 45(6):813–819. doi:10.1016/j.freeradbiomed.2008.06. 003 10. Baldeiras I, Santana I, Proenc¸a MT, Garrucho MH, Pascoal R, Rodrigues A, Duro D, Oliveira CR (2010) Oxidative damage and progression to Alzheimer’s disease in patients with mild cognitive impairment. J Alzheimers Dis 21(4):1165–1177 11. Guidi I, Galimberti D, Lonati S et al (2006) Oxidative imbalance in patients with mild cognitive impairment and Alzheimer’s disease. Neurobiol Aging 27(2):262–269. doi:10.1016/j.neuro biolaging.2005.01.001 12. Lo´pez N, Tormo C, De Blas I, Llinares I, Alom J (2013) Oxidative stress in Alzheimer’s disease and mild cognitive impairment with high sensitivity and specificity. J Alzheimers Dis 33(3):823–829. doi:10.3233/JAD-2012-121528 13. Cervellati C, Cremonini E, Bosi C, Magon S, Zurlo A, Bergamini CM, Zuliani G (2012) Systemic Oxidative Stress in Older Patients with Mild Cognitive Impairment or Late Onset Alzheimer’s Disease. Curr Alzheimer Res 14. Stocker R (2007) Vitamin E. Novartis Found Symp 282:77–87. doi:10.1002/9780470319444.ch6
15. Masaki KH, Losonczy KG, Izrnirlian G (2000) Association of vitamin E and C supplement use with cognitive function and dementia in elderly men. Neuroiem 54:1265–1272 16. Morris MC, Evans DA, Bienias JL, Tangney CC, Wilson RS (2002) Vitamin E and cognitive decline in older persons. Arch Newrol 59(7):1125–1132. doi:10.1001/archneur.59.7.1125 17. Engelhart MJ, Geerlings MI, Ruitenberg A (2002) Dietary intake of antioxidants and risk of Alzheimer disease. JAMA 287(24):3223–3229. doi:10.1001/jama.287.24.3223 18. Grodstein F, Chen J, Willett WC (2003) High-dose antioxidant supplements and cognitive function in community-dwelling elderly women. Am J Clin Nutr 77(4):975–984 19. Zandi PP, Anthony JC, Khachaturian AS et al (2004) Reduced risk of Alzheimer disease in users of antioxidant vitamin supplements: the cache county study. Arch Neurol 61(1):82–88. doi:10.1001/archneur.61.1.82 20. Isaac MG, Quinn R, Tabet N (2008) Vitamin E for Alzheimer’s disease and mild cognitive impairment. Cochrane database Syst Rev 16 (3):CD002854. doi: 10.1002/14651858.CD002854.pub2 21. Sano M, Ernesto C, Thomas RG et al (1997) 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(17):1216–1222. doi:10.1056/NEJM199704243361704 22. Petersen RC, Thomas RG, Grundman M et al (2005) Vitamin E and donepezil for the treatment of mild cognitive impairment. N Engl J Med 352(23):2379–2388. doi:10.1056/NEJMoa050151 23. Seyedian M, Falamh M, Nourouzian M, Nejat S, Delavar A, Ghasemzadeh HA (2008) Validity of the Farsi version of MiniMental State Examination. J Med Counc I.R.I. 25(4):408–414 (Abstract in English) 24. Yu LW, Latriano L, Duncan S, Hartwick RA, Witz G (1986) High-performance liquid chromatography analysis of the thiobarbituric acid adducts of malondialdehyde and trans, trans-muconaldehyde. Anal Biochem 156(2):326–333 25. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A (1993) A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci (lond) 84(4):407–412 26. Beutler E, Duron O, Kelly BM (1963) Improved method for the determination of blood glutathione. J Lab Clin Med 61:882–888 27. Morris MC, Beckett LA, Scherr PA, Hebert LE, Bennett DA, Field TS, Evans DA (1998) Vitamin E and vitamin C supplement use and risk of incident Alzheimer disease. Alzheimer Dis Assoc Disord 12(3):121–126. doi:10.1097/00002093-19980900000001 28. Sattler C, Toro P, Scho¨nknecht P, Schro¨der J (2012) Cognitive activity, education and socioeconomic status as preventive factors for mild cognitive impairment and Alzheimer’s disease. Psychiatry Res 196(1):90–95. doi:10.1016/j.psychres.2011.11.012 29. Letenneur L, Gilleron V, Commenges D, Helmer C, Orgogozo J, Dartigues J (1999) Are sex and educational level independent predictors of dementia and Alzheimer’s disease? Incidence data from the PAQUID project. J Neurol Neurosurg Psychiatry 66(2):177–183. doi:10.1136/jnnp.66.2.177 30. Scarmeas N, Luchsinger JA, Schup FN, Brickman AM, Cosentino S, Tang MX, Stern Y (2009) Physical activity, diet, and risk of Alzheimer disease. JAMA 302(6):627–637. doi:10.1001/jama 2009.1144 31. Buchman AS, Wilson RS, Bienias JL, Shah RC, Evans DA, Bennett DA (2005) Change in body mass index and risk of incident Alzheimer disease. Neurology 65(6):892–897. doi:10. 1212/01.wnl.0000176061.33817.90 32. Kukull WA, Higdon R, Bowen JD et al (2002) Dementia and Alzheimer disease incidence: a prospective cohort study. Arch Neurol 59(11):1737–1746. doi:10.1001/archneur.59.11.1737
123
Eur J Nutr 33. Kang JH, Cook N, Manson J, Buring JE, Grodstein F (2006) A randomized trial of vitamin E supplementation and cognitive function in women. Arch Intern Med 166(22):2462–2468. doi:10. 1001/archinte.166.22.2462 34. Luchsinger JA, Tang MX, Shea S, Mayeux R (2003) Antioxidant vitamin intake and risk of Alzheimer disease. Arch Neurol 60(2):203–208. doi:10.1001/archneur.60.2.203 35. Maxwell CJ, Hicks MS, Hogan DB, Basran J (2005) Supplemental use of antioxidant vitamins and subsequent risk of cognitive decline and dementia. Dement Geriatr Cogn Disord 20(1):45–51. doi:10.1159/000085074 36. Galasko DR, Peskind E, Clark CM et al (2012) Antioxidants for Alzheimer’disease randomized clinical trial with cerebrospinal fluid biomarker measures. Arch Neurol 69(7):836–841. doi:10. 1001/archneurol.2012.85 37. Miller ER 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E (2005) Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 142(1):37–46 38. Lock M, Loblaw A (2005) Vitamin E might increase risk of death. Can Fam Physician 51(6):829–831 39. Klein EA, Thompson IM, Tangen CM et al (2011) Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 306(14):1549–1556. doi:10. 1001/jama 2011.1437 40. Rautiainen S, Lindblad BE, Morgenstern R, Wolk A (2010) Vitamin C supplements and the risk of age-related cataract: a population-based prospective cohort study in women. Am J Clin Nutr 91(2):487–493. doi:10.3945/ajcn.2009.28528 41. Rathi S, Kern W, Lau K (2007) Vitamin C-induced hyperoxaluria causing reversible tubulointerstitial nephritis and chronic renal failure: a case report. J Med Case Rep 1:155. doi:10.1186/17521947-1-155 42. Farina N, Isaac MG, Clark AR, Rusted J, Tabet N (2012) Vitamin E for Alzheimer’s dementia and mild cognitive impairment. Cochrane Database of Systematic Reviews 11; CD002854. doi: 10.1002/14651858.CD002854.pub3 43. Stuerenburg HJ, Ganzer S, Mu¨ller-Thomsen T (2005) Plasma beta carotene in Alzheimer’s disease. Association with cerebrospinal fluid beta-amyloid 1-40, (Abeta40), beta-amyloid 1-42 (Abeta42) and total Tau. Neuro Endocrinol Lett 26(6):696–698 44. de Oliveira BF, Veloso CA, Nogueira-Machado JA, de Moraes EN, Santos RR, Cintra MT, Chaves MM (2012) Ascorbic acid, alpha-tocopherol, and beta-carotene reduce oxidative stress and proinflammatory cytokines in mononuclear cells of Alzheimer’s
123
45.
46.
47.
48. 49.
50.
51.
52.
53.
54.
55.
56.
disease patients. Nutr Neurosci. doi:10.1179/1476830512Y. 0000000019 Rinaldi P, Polidori MC, Metastasio A et al (2003) Plasma antioxidants are similarly depleted in mild cognitive impairment and in Alzheimer’s disease. Neurobiol Aging 24(7):915–919. doi:10. 1016/S0197-4580(03)00031-9 Arora A, Willhite CA, Liebler DC (2001) Interactions of betacarotene and cigarette smoke in human bronchial epithelial cells. Carcinogenesis 22(8):1173–1178. doi:10.1093/carcin/22.8.1173 Kontush A, Mann U, Arlt S, Ujeyl A, Lu¨hrs C, Mu¨ller-Thomsen T, Beisiegel U (2001) Influence of vitamin E and C supplementation on lipoprotein oxidation in patients with Alzheimer’s disease. Free Radic Biol Med 31(3):345–354. doi:10.1016/S08915849(01)00595-0 Giblin FJ (2000) Glutathione: a vital lens antioxidant. J Ocul Pharmacol Ther 16(2):121–135 Spiteller G (2001) Lipid peroxidation in aging and age-dependent diseases. Exp Gerontol 36(9):1425–1457. doi:10.1016/S05315565(01)00131-0 Pratico` D, Clark CM, Liun F, Rokach J, Lee VY, Trojanowski JQ (2002) Increase of brain oxidative stress in mild cognitive impairment: a possible predictor of Alzheimer disease. Arch Neurol 59(6):972–976. doi:10.1001/archneur.59.6.972 Montine KS, Quinn JF, Zhang J, Fessel JP, Roberts LJ, Morrow JD, Montin TJ (2004) Isoprostanes and related products of lipid peroxidation in neurodegenerative diseases. Chem Phys Lipids 128:117–124. doi:10.1016/j.chemphyslip.2003.10.010 Keller JN, Schmitt FA, Scheff SW, Ding Q, Chen Q, Butterfield DA, Markesbery WR (2005) Evidence of increased oxidative damage in subjects with mild cognitive impairment. Neurology 64(7):1152–1156. doi:10.1212/01.WNL.0000156156.13641.BA Markesbery WR, Lovell MA (2007) Damage to lipids, proteins, DNA, and RNA in mild cognitive impairment. Arch Neurol 64(7):954–956. doi:10.1001/archneur.64.7.954 Rath M (2003) External and inherited cardiovascular risks: in: Rath M, 4th eds. Why animals don’t get heart attacks, published by Dr. Rath Health Foundation Husebye E, Engedal K (1992) The patterns of motility are maintained in the human small intestine throughout the process of aging. Scand J Gastroenterol 27(5):397–404. doi:10.3109/ 00365529209000095 Lloret A, Badı´a MC, Mora NJ, Pallardo´ FV, Alonso MD, Vin˜a J (2009) Vitamin E paradox in Alzheimer’s disease: it does not prevent loss of cognition and may even be detrimental. J Alzheimers Dis 17(1):143–149
