Alzheimer's disease (AD) is characterised clinically by progressive loss of memory and cognition, pathologically by senile plaques, neurofibrillary tangles and synapse loss(Reference Butterfield and Lauderback1). Oxidative stress plays a central role in this disease, and it is the first event that precedes the disease, and it is manifested by increase in protein oxidation, lipid peroxidation, DNA and mRNA oxidation and formation of reactive oxygen species (ROS) and reactive nitrogen species in the brain(Reference Lovell and Markesbery2).
The nervous system is particularly vulnerable to the deleterious effects of ROS and reactive nitrogen species, since it has the highest amount of oxygen to produce energy, and the brain contains high concentrations of PUFA that are highly susceptible to lipid peroxidation(Reference Mariani, Polidori and Cherubini3). Indeed, senile plaques and neurofibrillary tangles are directly associated with oxidative damage in AD, since this peptides can produce ROS(Reference Chauhan and Chauhan4, Reference Crouch, Harding and White5).
Cells have mechanisms to prevent or repair oxidative damage caused by ROS and reactive nitrogen species. These include antioxidant molecules and enzymes, such as glutathione peroxidase(Reference Crouch, Harding and White5). However, the brain has a relatively deficient antioxidant system, which contributes to its susceptibility to oxidative damage(Reference Mariani, Polidori and Cherubini3).
Se is an essential nutrient in the diet due to the requirement for selenocysteine in some selenoproteins. This trace element is known to provide protection from ROS-induced cell damage, and the proposed mechanisms mainly invoke the functions of glutathione peroxidase family and selenoprotein P(Reference Chen and Berry6).
Although some studies have shown the increase of oxidative stress in AD patients, there is a lack of information about the importance of Se as part of antioxidant enzymes in this disease. In this context, the present study aimed to evaluate nutritional status of Se in AD patients.
Methods
Subjects
Twenty-eight (eleven male and seventeen female) elderly diagnosed with probable AD (AD group) according to the National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer's Disease and Related Disorders Association criteria(Reference McKhann, Drachman and Folstein7) were included in the present study. These patients attended at the Geriatric Cognitive Ambulatory or at the Center of Reference in Cognitive Disorders, both at the Hospital das Clínicas of the São Paulo University Medical School (São Paulo, Brazil).
Twenty-nine (ten male and nineteen female) healthy volunteer elderly with normal cognitive function, mini-mental state examination ( ≥ 20)(Reference Folstein, Folstein and McHugh8, Reference Seabra, Concílio and Villarea9) sex-matched with those in the AD group, who attended at the Multidisciplinary Group of Assistance to the Aged Ambulatory at the Hospital das Clínicas of the São Paulo University Medical School (São Paulo, Brazil), were included in the control group (C group).
Participants were selected for the study according to the following inclusion parameters: aged 60 years or older; absence of acute inflammation, infection, fever, diarrhoea, cancer, diabetes, autoimmune disease and vitamin and mineral supplement intake. AD patients should have an active caregiver.
The present study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects/patients were approved by Ethics Committee of the Faculty of Pharmaceutical Sciences at the University of São Paulo (no. 455) and by the Ethics Committee of the Hospital das Clínicas of the University of São Paulo Medical School (no. 0710/08). Written informed consent was obtained from participants of the C group and caregivers of AD patients.
Selenium intake
Se intake was evaluated by using a 3-d (2 weekdays and 1 weekend day) dietary food record, up to 7 d before the blood sample was drawn. AD patients and their caregivers were requested to register what they had eaten. Se intake was measured by using NutWin software (version 2.5; EPM-UNIFESP, São Paulo, Brazil).
Software database was supplied with Se data from the study of Ferreira et al. (Reference Ferreira, Gomes and Bellato10). Items consumed by the participants, which were not originally listed in this database, were included in the software as data obtained from food composition tables(Reference Philippi11) or food labels provided by participants.
Se intake was adjusted by the energy, according to Willet(Reference Willet12), using linear regression (linear regression of nutrient intake on total energy intake) and addition of a constant (mean energetic intake of the group). Se intake was compared with dietary reference intake recommendations for the particular age and sex group(13).
Biochemical assays
Se concentration was determinate in plasma, erythrocyte and nail samples by using hydride generation atomic absorption spectroscopy(Reference Gonzaga14).
Plasma and erythrocytes were obtained from a fasting, morning blood sample. Toenails and fingernails samples were collected by the participants after 20 d of nails growth without nail polish, up to 20 d before the blood sample was drawn.
All reagents had analytical grade or higher purity from Merck. Nanopure water was used to prepare all solutions and to dilute the samples.
The standard reference material Seronorm® was analysed for testing the accuracy and precision of the analytical technique.
Statistical analysis
All statistical analyses were carried out using the Statistical Package for the Social Sciences software, version 17.0, for Windows (SPSS, Chicago, IL, USA).
The results were showed as means and standard deviations.
Variables distribution was evaluated by using Kolmogorov–Smirnov test. Differences between AD and C groups were analysed with Student's t test. Pearson's correlation coefficients were used to estimate correlations between nutritional parameters and between cognitive evaluation and nutritional status.
A P value of 0·05 was considered statistically significant.
Results
In the present study, twenty-eight elderly (39·3 % men and 60·7 % women) in the AD group and twenty-nine healthy elderly (34·5 % men and 65·5 % women) in the C group were evaluated. There was no sex difference between the two groups; however, the mean age of the AD group was 80·6 (sd 5·7) years, statistically different from the C group, which showed a mean age of 71·2 (sd 6·2) years (P < 0·05). Therefore, given that age might influence Se concentrations, correlation analysis between Se parameters and age was performed, but correlations were not observed (P>0·05).
Se levels in plasma and erythrocyte were significantly higher in the C group, although in both groups some participants showed values below the normal range(Reference Ortuño, Ros and Periago15). There is no normal values range for Se level in nails, but higher values in the C group were observed when compared with the AD group (Table 1).
Selenium concentrations in plasma (μg/l), erythrocytes (μg/l) and nails (μg/g), and selenium content in diet (μg/d) of elderly with Alzheimer's disease (AD) and those in the control (C) group
* P < 0·005.
Higher Se intake was observed in the C group, and it was adequate in 38·5 % of the AD group and in 63 % of the C group (Table 1).
Discussion
Deficient Se intake was observed in the most of the AD patients, although the dietary reference intake recommendation was only partially achieved by the C group. Since Se content of foods is largely related to the Se content of soil, it is important to reveal that in São Paulo state there is low Se concentration in the soil, and thus high deficiency of this mineral is observed in this population(Reference Cozzolino16).
Assessment of Se intake from the diet has many difficulties, namely the absence of specific food composition tables for this trace element and Se variation in different regions(Reference Combs17–Reference Al-Saleh, El-Doush and Billedo19). This is probably the main reason for the lack of studies describing in detail the Se intake in AD patients.
Assessment of Se levels in blood is a useful biomarker of Se status. Plasma is a marker of current exposure, while erythrocytes reflect longer-term nutritional status, due to its incorporation in erythrocyte synthesis, which have a half-life of 120 d(Reference Thomson20, Reference Navarro-Alarcon and Cabrera-Vique21). Nail clippings are considered a superior marker of Se status because they provide a time-integrated measure of exposure of up to a year(Reference Satia, King and Morris22). Some authors suggest that Se status should be assessed by determination of two or more biomarkers, in order to avoid possible misunderstandings arising from the hierarchy of importance of selenoproteins(Reference Thomson20, Reference Slotnick and Nriagu23). According to these authors, our assessment of Se status covered different periods of exposure, allowing us to verify Se intake in different periods.
There is a lack of studies correlating Se status and AD, and most of them only assessed Se levels in plasma or whole blood. The studies about Se in AD patients are inconclusive. Ceballos-Picot et al. (Reference Ceballos-Picot, Merad-Boudia and Nicole24) observed higher Se plasma and erythrocyte levels in AD patients when compared with the control group. However, Smorgon et al. (Reference Smorgon, Mari and Atti25) assessed the association between micronutrients and cognitive function, and observed that AD patients showed lower plasma Se when compared with control subjects. Other studies noticed that Se levels were positively correlated with cognitive function in elderly subjects, and thus Se deficiency could be a risk factor for AD(Reference Berr, Balansard and Arnaud26–Reference Akbaraly, Hininger-Favier and Carrière28). Although the average plasma and erythrocyte Se concentrations in the AD and C groups were below the recommended levels, individual values were significantly different in each group, revealing that AD has an important role in Se deficiency. Confirming these data, Se level in nails was significantly lower in the AD patients when compared with control subjects.
Some studies have shown that Se status decreases slightly in elderly compared with younger adults(Reference Chen, Lai and Wu29–Reference Letsiou, Nomikos and Panagiotakos31). It can reflect lower bioavailability, increased requirements, metabolic changes, or a diet limited in energy which may not be sufficiently nutrient dense to provide adequate levels of micronutrients(Reference Arnaud, Akbaralyc and Hininger30, Reference Planas, Conde and Audivert32). In the present study, no significant correlation between Se parameters and age was observed. However, the age difference between the AD and C groups could be a bias in the present study, since nutritional status of Se can decrease with age. Indeed, the present study has a small sample size, and this can limit the interpretation of our data.
The brain is the last organ to be depleted in Se deficiency and, in repletion, it is the first one to establish adequate levels of Se. This preferential treatment suggests the importance of this antioxidant mineral in brain functioning, since it is a main constituent of selenoproteins(Reference Benton33). Glutathione peroxidase is an essential line of defence against free radicals acting against hydrogen peroxide and lipid peroxidation, protecting the brain against oxidative stress, which has a central role in AD. Besides, selenoprotein P, which is synthesised at the cerebral level and protects the brain against oxidative damage, is the most important selenoprotein for cerebral functions(Reference Schweizer, Schomburg and Savaskan34, Reference Crack, Cimdins and Ali35). Thus, Se may affect the rate of disease via protection against ROS, directly as an antioxidant or indirectly by improving metabolism(Reference Staehelin36).
Oxidative stress has an important role in AD aetiology, and it is the earliest event preceding this disease, although some structures formed in AD are related to formation of ROS(Reference Zhu, Lee and Perry37). Thus, the relationship between Se and AD could have two meanings: Se could be depleted owing to the oxidation that accompanies ageing and AD progression; and change in Se levels could be the first event in relation to dietary intake(Reference Luchsinger and Mayeux38). However, as we did not evaluate oxidative stress in the present patients, we cannot establish direct association between Se deficiency and oxidative stress in AD patients. In this way, more studies with larger population are needed in order to find the exactly relationship between Se and AD, since a limitation of the present study was the small number of participants.
Conclusions
AD patients showed lower Se levels, although a Se deficiency was also observed in the C group. Thus, we can suggest that the oxidative stress present in AD has an association with Se deficiency observed in the present AD patients. In order to overcome the problem of Se deficiency in AD patients, new strategies should be developed.
Acknowledgements
The work was generously supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). The authors made the following contributions to the study: B. R. C., S. M. F. C. and T. P. O. did the study concept and design, analysis and interpretation of data; B. R. C. did the preparation of the manuscript; W. J.-F. and O. J. did the subject recruitment and review of the manuscript; M. I. D. F. did the cognitive function assessment and review of the manuscript. The authors indicate that they have no financial relationships relevant to the present article to disclose.
