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INTRODUCTION
The most common form of presentation of all dementias is Alzheimer's disease (AD), characterized by a progressive neurodegenerative process that affects large areas of the cerebral cortex and hippocampus. Abnormalities are generally detected first in brain tissue involving the frontal and temporal lobes, subsequently progressing to other areas of the neocortex at rates that vary considerably between individuals. The characteristic pathological findings of AD are accumulation of amyloid β peptide (Aβ) in neuritic plaques, hyperphosphorylation of tau protein, and degeneration of neurons in brain regions such as the hippocampus, resulting in progressive cognitive dysfunction (1). With regard to the underlying alterations of the disease, participation by oxidative damage caused by free radicals has been demonstrated. The brain is particularly vulnerable to oxidative stress because it contains abundant polyunsaturated fatty acids; in addition, it consumes more oxygen per gram compared to other tissues and has less antioxidant capacity. The initial contribution of oxidative stress in Alzheimer's disease is demonstrated by oxidative modifications of lipids, proteins, and nucleic acids in the brain of these patients, as well as in cellular and animal models of AD. Likewise, βA has been shown to induce membrane lipid oxidation (2).
Epidemiological evidence suggests that people with a high intake of antioxidants in their diet have lower rates of AD (3,4). Similarly, some clinical trials have reported beneficial effects in older people with mild cognitive impairment with supplementation with long chain omega-3 polyunsaturated fatty acids (PuFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (5). Furthermore, a cohort study in older men and women reported that higher serum concentrations of PuFAs were significantly associated with better patient performance on neuropsychological test. Elevated levels of EPA + DHA are significantly associated with a lower risk of dementia (6). In a meta-analysis, a beneficial effect of the consumption of these fatty acids was demonstrated in people with mild cognitive impairment in the subdomains of attention and processing speed (7). PuFAs are mainly incorporated into phospholipids, sphingolipids and plasmalogens and contribute to modulate the biophysical properties of cell membranes (fluidity, shape and thickness, permeability) and the activity of transmembrane proteins (8). Doses of between 2 and 6 g of DHA have shown incorporation into plasma phospholipids and this is increased in a dose-dependent manner (9). Previous data of our laboratory shows that dietary supplementation with fish oil has the following beneficial effects in patients diagnosed with probable Alzheimer's disease: it decreases plasma levels of lipoperoxides and nitric oxide catabolites and increases the ratio of reduced glutathione/oxidized glutathione. These changes are in parallel with the decrease in the ratio of omega 6 fatty acids/omega 3 fatty acids in erythrocytes (10). In the present study, the efficacy of omega 3 supplementation was evaluated in these patients by assessing the levels of proteins oxidation, hydroperoxides and enzymatic activities of catalase and superoxide dismutase.
MATERIALS AND METHODS
STUDY DESIGN
A randomized, double-blind clinical trial was conducted at the Cognitive Impairment Clinic of the Neurology Department of the Western National Medical Center, Mexican Social Security Institute in Guadalajara, Jalisco, Mexico. This study was conducted in accordance with the updated Declaration of Helsinki and all procedures were approved by the Ethics and Health Research Committee of the Mexican Social Security Institute (protocol number: 2014-785-011).
The diagnosis of AD was made by: a) a Mini Mental State Assessment (MMSE) (11) performed during the first visit, with a score of less than 25 for literate people and 20 for non-literate people; 2) DSM-V criteria for dementia (12); and 3) NINCDS-ADRDA diagnostic criteria (13). The patients had at least 2.5 years of history of cognitive decline. The criteria for excluding patients were: consumption of antioxidant supplements, history of acute kidney or liver dysfunction, tobacco, drug or alcohol abuse, intolerance, contraindication or allergy to fish oil. Twenty patients diagnosed with probable AD were randomized 1:1 to receive oral fish oil or placebo, with a computer-generated randomization sequence. Fish oil and placebo were administered in gel dosage form and to ensure masking the gels were identical in appearance, packaging, and labeling. Randomization and allocation were concealed from investigators and patients until the end of the study. Identification numbers were assigned to ensure patient confidentiality. Informed consent was obtained from the patient and the caregiver. Patients took one gel orally per day. The fish oil gels contained 0.45 g of EPA and 1 g of DHA. Participants reported daily consumption of the supplement on a consumption record sheet. The rate of adherence to treatment was greater than 80 %.
OXIDATIVE STRESS MARKERS ASSESSMENT
At the beginning of treatment and every six months a blood sample was drawn from the patients to evaluate oxidative stress markers. Blood samples were collected in tubes with 0.1 % ethylenediaminetetraacetic acid (EDTA) and the plasma was separated by centrifugation at 2,000 rpm for ten minutes at room temperature. The samples were stored at -80 °C degrees until use. All optical density readings were made with a uV/VIS spectrophotometer (Benchmark™ Plus Microplate from BioRad; Hercules, CA, USA).
The levels of carbonyl groups in proteins were quantified according to Lenz AG et al. (14) with minor modifications as follows: to 0.2 ml of plasma 1 ml of 10 mM 2,4-dinitrophenylhydrazine was added dissolved in 2M HCl. The samples were incubated for one hour at room temperature and 333 µl of trichloroacetic acid (30 %, w/v) were added. Then, the suspension was centrifuged at 14,000 rpm for 20 minutes. The resulting precipitate was washed three times with 1 ml of ethyl acetate ethanol solution (1:1). The supernatant was discarded and the final pellet was dissolved with 1 ml of 6M guanidine hydrochloride and incubated for 15 minutes at room temperature. The absorbance of the samples was read at 370 nm.
Hydroperoxide quantitation was determined with 0.1 ml of plasma mixed with 0.9 ml of reaction medium (100 mM xylene orange, 250 mM ferrous ammonium sulfate, 25 mM sulfuric acid, and 4 mM butylhydroxytoluene in 90 % methanol). The samples were incubated for 30 minutes at room temperature and then centrifuged at 10,000 rpm for five minutes. The absorbance of the supernatant was read at 560 nm. A standard curve was run with known concentrations of hydrogen peroxide (15).
Catalase activity was assessed with 0.1 ml of plasma mixed with 0.9 ml of reaction medium (65 mM hydrogen peroxide in 60 mM phosphate buffer, pH = 7.4) and incubated at 37 °C for two minutes. The reaction was stopped with 2 ml of 32.4 mM ammonium molybdate. The absorbance of the samples was recorded at 374 nm to quantify the hydrogen peroxide remaining in the reaction (16).
Superoxide dismutase activity was quantified using a reagent kit (No. 706002, Cayman Chemical Company®, USA). The manufacturer's instructions were followed for the detection of superoxide anion generated by the enzyme xanthine oxidase and hypoxanthine by reaction with tetrazolium salt. Plasma samples were diluted 1:5 in sample buffer and 0.2 ml of the radical detector was added to 10 µl of diluted plasma. After slow stirring, 20 µl of xanthine oxidase was added and the samples were incubated for 20 minutes at room temperature and the absorbance was read at a wavelength of 440 nm. Superoxide dismutase activity is reported in u/ml.
STATISTICAL ANALYSIS
Data were analyzed as mean values ± standard deviation. The differences in the parameters studied between the groups were evaluated by analysis of variance (ANOVA) and the Mann-Whitney u test. A p value < 0.05 was considered as statistically significant. The analyzes were performed in SPSS version 21.
RESULTS
CHARACTERISTICS OF THE STuDY POPuLATION
Of a total of 87 AD patients treated at the Neurology Department's Cognitive Impairment Clinic, 20 met the inclusion criteria and were randomly assigned to receive fish oil (ten patients) or placebo (ten patients). At the end of the intervention, 17 patients completed the one-year trial. In the fish oil group, two patients withdrew from the study, one due to lack of adherence to treatment and the other due to intolerance to the taste of fish. In the placebo group, one patient discontinued their participation in the study due to the development of high blood pressure.
OXIDATIVE STRESS MARKERS
Figures 1A and 1B exhibit that plasma levels of carbonyl groups in proteins and hydroperoxides, respectively, have similar values in both treatment groups at the beginning of the study. However, at six and 12 months the values decreased significantly in the fish oil group, while in the group treated with placebo no changes were observed in both oxidative stress markers at six and 12 months.
Figure 1. Plasma levels of biochemical markers at the beginning and after six and 12 months of treatment in the placebo (P) and fish oil (F) group. Levels of carbonyl groups in proteins (A), hydroperoxides (B), catalase activity (C) and superoxide dismutase activity (D). Data are expressed as mean ± standard deviation. The comparison was calculated with the Mann-Whitney u test. *p < 0.05.
The enzymatic activities of catalase and superoxide dismutase (Figures 1C and 1D, respectively) in plasma at the start of treatment were similar in both study groups. Catalase activity increased significantly at six and 12 months after treatment only in patients treated with fish oil. While the superoxide dismutase activity is not modified in neither of the two study groups.
DISCUSSION
Oxidative stress plays an important role in the pathogenesis of AD. In the present study, it was found that patients who consume PuFAs at a stable dose of DHA and EPA show decreased oxidation of plasma proteins and lipids in addition to an increase in catalase activity. This beneficial effect may be important, since the increase in production of reactive oxygen species (ROS) associated with AD affects mitochondrial activity, metal homeostasis and the deterioration of antioxidant defense, which directly affects synaptic activity and neurotransmission, which leads to cognitive deterioration. Abnormal cellular metabolism in AD affects the production and accumulation of amyloid and hyperphosphorylated tau protein, which could independently exacerbate mitochondrial dysfunction and contribute to ROS production, thus preserving a vicious cycle to the detriment of cognitive function (17).
Recent evidence suggests that the actions derived from PuFAs are mediated through oxidative and non-oxidative metabolic pathways that convert them to bioactive lipid metabolites. One of the non-oxidative pathways involves the conversion of DHA and EPA to the endocannabinoids docosahexaenoyl ethanolamide and eicosapentaenoyl ethanolamide, which have similar effects to those of Δ9-tetrahydrocannabinol. Endocannabinoids play important physiological roles that are mainly exerted through activation of the cannabinoid receptor-1 (CB1) and -2 (CB2). CB1 is found predominantly in the CNS, and CB2 is found in peripheral immune cells. DHEA has anticancer, anti-inflammatory, and synaptogenic properties (18). Additionally, the following effects of omega 3 fatty acids have been demonstrated: a) their anti-inflammatory effect, which is based on the down regulation of nuclear factor kB (19); b) the production of oxygenated metabolites of EPA and DHA that actively promote the resolution of inflammation, such as resolvins and protectins (20); and c) its effect on modifying the biophysical properties of the membranes resulting in changes in the activity of ion channels, receptors and the binding between phospholipids and proteins, which may be important, due to the nature of the interaction of the membranes with the β-amyloid peptide (21).
DHA has also been shown to decrease β-amyloid peptide levels, regulate the activity of anti-apoptotic genes such as Bcl-2, BCl-xl, and Bfl-1 (22), and modulate the phosphatidyl inositol 3-kinase-Akt pathway (23). Similarly, DHA augments angiongenesis and neurogenesis in an in vivo model (24). On the other hand, with respect to the estimated half-life of DHA in the brain, it has been identified that it is approximately 2.5 years, its bioavailability begins with ingestion from which it is incorporated very quickly into the membrane phospholipids of many tissues and relatively little time is required to observe its effects (25). The incorporation of DHA into cholesterol esters reflects the consumption of DHA in the previous 1-2 weeks. In erythrocyte membranes, its consumption is reflected in the previous 1-2 months and in adipose tissue from a period of previous years of consumption (26). Measurements of DHA levels in erythrocytes and plasma have been correlated with its concentration in the brain, retina, and liver. Stopping the intake of DHA as a supplement RESULTS in a rapid release of this lipid from the plasma membrane. This shows that the constant consumption of DHA is necessary to maintain its effect on the lipid rafts of the plasma membranes (9).
Consistent with the above, it is known that DHA increases the binding of soluble oligomers of amyloid β peptide to membrane phospholipids and this has a beneficial effect (27). Therefore, elevated levels of EPA and DHA in the plasma membrane can affect the metabolism of amyloid β peptide and reduce the risk or progression of Alzheimer's disease. Additionally, in this work it was found that the increase in the content of EPA and DHA in erythrocytes is associated with an improvement in the fluidity of the membranes. This can be attributed to the fact that EPA and DHA exert some of their metabolic functions as part of the structure of the phospholipids that make up cell membranes, particularly phosphatidylcholine and phosphatidylserine. Due to their high degree of polyunsaturation, these fatty acids provide fluidity to the membranes, an essential characteristic that allows the mobility of proteins, either on the surface or inside the lipid bilayer (28).
