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Journal of Nutritional Biochemistry 24 (2013) 803 808

The role of long chain omega-3 polyunsaturated fatty acids in reducing lipid peroxidation among elderly patients with mild cognitive impairment: a case-control study
Lai Kuan Lee a, b, Suzana Shahar c,, NorFadilah Rajab d , Noor Aini Mohd Yusoff e, Rahman A. Jamal f , Sue Mian Then f
Nutrition Science Programme, School of Healthcare Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia b Nutrition Programme, School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia c Dietetics Programme, School of Healthcare Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia d Biomedical Science Programme, School of Diagnostics & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia e Faculty of Therapeutic Sciences, Masterskill University College of Health Sciences, Jalan Kemacahaya, 43200 Selangor Darul Ehsan, Malaysia f Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latiff, Bandar TunRazak, 56000 Kuala Lumpur, Malaysia
a

Received 1 August 2011; received in revised form 25 April 2012; accepted 26 April 2012

Abstract The present work explores the effect of dietary omega-3 polyunsaturated fatty acids (PUFAs) intake on lipid peroxidation among mild cognitive impairment (MCI) patients. The plasma lipid hydroperoxide (LPO) levels in 67 MCI patients were compared to those of 134 healthy elderly controls. Omega-3 PUFA intake was assessed using an interviewer-administered food frequency questionnaire. Apolipoprotein E genotyping was performed using polymerase chain reaction and restriction enzyme digestion. The association between various confounders and lipid peroxidation was evaluated using regression analysis. The influence of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) intake on LPO level was investigated. The results revealed that LPO levels were significantly higher in the MCI group than in the control group. Inverse correlations were found between DHA and EPA intake and LPO level among the MCI group. LPO levels decreased significantly with increasing DHA and EPA intake. In summary, the findings revealed that DHA and EPA can play a role in alleviating oxidative stress and reducing the risk of neurodegenerative diseases. 2013 Elsevier Inc. All rights reserved. Keywords: Docosahexaenoic acid; Eicosapentaenoic acid; Mild cognitive impairment; Lipid peroxidation; Polyunsaturated fatty acids

1. Introduction Mild cognitive impairment (MCI) is the unique transitional state in which slight alterations in memory or other cognitive abilities coexist with predominantly normal activities of daily living (ADL) and absence of dementia [1]. Researcher have consistently found that almost 50% of MCI individuals progress to develop Alzheimer's disease (AD) within 4 years [2], indicating that MCI is prodromal stage of AD. In recent decades, increased oxidative stress during aging has been proposed as a contributor to AD, which suggests that excessive oxidative damage may occur in MCI patients. According to the free radical theory, oxidative stress is a complication of a progressive accumulation of oxidative damage from hazardous biomolecules that results from imbalances between
Funding source: This work was supported by the Universiti Kebangsaan Malaysia under the Research University (Project code: UKM-GUP-SK07-21-041) grant scheme. Corresponding author. Tel.: + 60 392897194; fax: + 60 326938717. E-mail address: suzana.shahar@gmail.com (S. Shahar).

pro-oxidants and antioxidants [3]. Accumulating evidence has also suggested elevated levels of oxidative and nitrosative damage and lipid peroxidation in AD and MCI patients [46]. Lipid peroxidation generates a variety of relatively stable end products [mainly aldehyde by-products, such as malondialdehyde (MDA) and more reactive --unsaturated reactive aldehydes, such as 4-hydroxy-2-nonenal (HNE) and 2-propenal (acrolein)]. Evidence suggests that these byproducts may be capable of forming adducts with DNA and proteins that accelerate the decline in normal brain function [7]. Lipid peroxidation in plasma may alter biological properties (principally the degree of membrane uidity) and lead to inactivation of membrane-bound receptors or enzymes [8]. Therefore, it is important for researchers to look for potential therapeutic targets that can delay the progression of oxidative damage in the early stages of MCI. For this reason, increasing attention has been paid to the role dietary nutrients may play in reducing oxidative stress. In particular, recent studies have revealed that omega-3 polyunsaturated fatty acids (PUFAs) are more susceptibility to oxidation and involved in lipid metabolism [9,10], with docosahexaenoic acid (DHA) being the most oxidisable omega-3

0955-2863/$ - see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2012.04.014

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PUFA[11], as it is the most unsaturated. However, in vitro [12] and in vivo [13] studies have suggested that the relation between chemical structure and susceptibility to oxidation is not as theoretical considerations might suggest. On the contrary, Richard et al. [11] have reported that long chain PUFAs may indirectly act as antioxidants by lowering reactive oxygen species (ROS) production and superoxide scavenging, while reduced lipid peroxidation has been observed after supplementation with omega-3 PUFAs [14]. These ndings have stimulated considerable research interesting establishing other well-known dietary antioxidants (such as vitamin C) as leading alternatives to the antioxidant properties of omega-3 PUFAs. Epidemiological studies have reported that AD risk was inversely associated with PUFA intake [15,16]. However, the impact of omega-3 PUFA intake on the lipid peroxidation status of elderly MCI patients is largely unknown; therefore, this impact is the subject of the current investigation. The aim of the present study was to evaluate the effects of dietary long chain omega-3 PUFA intake on lipid peroxidation, as assessed by lipid hydroperoxide (LPO) levels, in elderly MCI patients by comparing them to the effects on age, gender and ethnicity matched control.
2. Materials and methods 2.1. Recruitment of subjects This case control study involving community-dwelling elderly individuals who were recruited from 15 public residential buildings in central Cheras, a metropolitan city of Kuala Lumpur, Malaysia. The inclusion criteria were age 60 or older, no known physical or mental illness and ability to communicate. The exclusion criteria were a diagnosed psychiatric disorder or an untreatable chronic disease, such as cancer, kidney failure, coronary heart disease and uncontrolled diabetes. Chronological age (as indicated by identity cards), educational attainment (dened as the number of years of schooling), ethnicity and gender were obtained during the interview session. The Leicester Height Measure (CMS Weighing Equipment, United Kingdom) was used to measure standing height to the nearest 0.1 cm. Body weight was measured to the nearest 0.1 kg using a calibrated digital bathroom scale (with a100 g lithium battery) (Tanita HD-319, Japan). Body mass index (BMI) was calculated as body weight in kilograms divided by squared standing height in meters. Informed consent for participation in the study was obtained from each subject and the study protocol was approved by the Universiti Kebangsaan Malaysia Medical Research Secretariat. 2.2. MCI diagnosis and control group This study adopted frequently used Mayo clinic MCI diagnostic criteria [17]. In depth description of these criteria have been published elsewhere [18]. Briey, the subjects were diagnosed as having MCI if they met the following conditions: (1) memory complaints, (2) preserved global cognitive function, (3) intact ADL and instrumental ADL, (4) not demented, (5) not depressed, and (6) abnormal cognitive impairments for age. Once 67 elderly MCI patients had been identied, 134 age-, gender- and ethnicitymatched healthy elderly individuals (who also underwent neuropsychological evaluation) were selected as the control group. 2.3. Neuropsychological measures The Malay version of the Mini-Mental State Examination (MMSE) [19] was administered to measure global cognitive function. The Dementia Rating Scale (DRS) [20] was used as dementia screening tool. The Rey Auditory Verbal Learning Test (RAVLT)[21] was used to test verbal learning and memory. The total number of words learned in ve trials (total immediate recall) and delayed recall (following a 15-minute delay) were also examined. Short-term memory and attention were assessed using the Digit Span subtest of the Wechsler Adult Intelligence Scale [22]. Visual construction and abstract thinking skills were measured by the Clock Drawing Test (CDT)[23]. All of the neuropsychological measures were administered by clinical psychologists and were scored according to their published protocols. 2.4. Dietary assessments Long chain omega-3 PUFA intake was assessed using an interviewer-administered validated food frequency questionnaire (FFQ). This specied semi-quantitative FFQ was designed to determine omega-3 PUFAs intake over the previous year among the multiethnic population of Malaysia. The 45-item FFQ was used with food models, a food atlas and food booklets to determine consumption frequencies for each type of food. Specic food items were grouped into six categories (sh and seafood, meat and poultry, egg and eggs products, vegetables, cooking oils and dairy products), and the

amount consumed from each category was recorded. Detailed information on brands, cooking methods, parts of food consumed and fortied foods were included to avoid under-reporting. Open-ended questions were used to assist the participants. When quantitative data could not be provided, it was estimated by a normal serving size. The average daily omega-3 PUFA intake was analysed using the Nutrition Data System for Research (NDSR 2009) software and standard serving sizes of the food items were determined by the Malaysian Food Composition Table [24]. Additional information on nutritional labelling for processed canned food, fortied foods and self-designed recipes was gathered. The total dietary eicosapentaenoic acid (EPA) and DHA intakes were determined by the multiplying food intake (in g/day) by the EPA and DHA contents of the foods (in mg/g edible portion). The total energy intake for the previous week was determined using the FoodWorks nutrient analysis software package (version 3.02; Xyris software) [25]. 2.5. Blood sampling Peripheral venous blood sampling was performed after a 10-h overnight fast. A total of 20 ml of venous blood was drawn into a serum separating tube and centrifuged at 5C for 10 min at 1200 g. The serum isolation and total cholesterol (TC), triglyceride (Tg), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) measurements were conducted within 2 h of taking the sample. In addition, 5 ml of fasting peripheral venous blood was drawn into EDTA tubes, centrifuged at 3000 rpm to obtain plasma and stored at 80C for analyses within 3 months of the blood collection. 2.6. Measurement of LPO level The plasma LPO levels were measured using the Calbiochem Lipid Hydroperoxide Assay Kit (Merck, Germany) according to the manufacturer's instructions. This assay kit uses redox reactions with ferrous ions to directly measure LPO. Hydroperoxides molecules are highly unstable and react with ferrous ions to produce ferric ions. The resulting ferric ions are detected using thiocyanate ion as the chromogen. This quantitative extraction method extracts LPO in chloroform, thus eliminating any interference caused by hydrogen peroxide or endogenous ferric ions in the plasma and providing a sensitive and reliable assay for lipid peroxidation. An LPO standard was prepared using the reagents provided (including extract R, triphenylphosphine, chromogen, chloroform, methanol and a mixture of chloroform and methanol). Each endpoint supernatant was monitored by absorbance at 500 nm. The signal was read against the standard curve, and the results were expressed as M. 2.7. Determination of apolipoprotein E (APOE) genotypes Genomic DNA was isolated from leucocytes using the QIAmp DNA Blood Mini Kit (Qiagen, Crawley, United Kingdom). APOE genetic polymorphisms were determined using a standard protocol [26]. Briey, the APOE gene was amplied by a polymerase chain reaction (PCR) in a MyCycler machine programmed for initial denaturation at 95C for 4 min, followed by 30 cycles of denaturation at 94C for 1 minute, annealing at 58C for 1 minute, extension at 72C for 30 s, a nal extension at 72C for 10 min and a holding period in the machine at 4C. Each amplication reaction contained 0.1 M of F4 primer, 0.1 M of F6 primer, 12.5 l of Master Mix 2X, approximately 50 g of DNA and MiliQ water to reach a nal volume of 25 l. After the PCR amplication, 13.1 l of the PCR products was digested overnight by 8 units of HhaI enzyme and 1.5 l of NE4 buffer. The digested product was then loaded onto a 12% polyacrylamide gel and electrophoresed for 70 min at 115 V. After the electrophoresis, the gel was treated with ethidium bromide for 5 min and visualised under ultraviolet reection and transillumination. The APOE isoforms were visualised, identied and differentiated according to known base pair fragment size. The subjects were categorised as carriers or non-carriers of APOE4. 2.8. Statistical analysis The independent-sample Student's t and 2 tests were used to compare the characteristics of the MCI group with those of the control groups. The subjects with the 2/2 and 3/3 APOE genotypes were pooled due to the small number of 2/2 subjects. Similarly, the 2/4, 3/4 and 4/4 subjects were pooled due to the small number of 4/4 subjects. Pearson correlation coefcients were calculated for long chain omega-3 PUFA intake and cognitive performance and for LPO level and cognitive performance (whole study population). A simple regression analysis was used to investigate the possible predictors of plasma LPO level in the MCI and control groups. The modulating effects of long chain omega-3 PUFAs on LPO levels was assessed using the generalised linear model (GLM). We rst evaluated the individual effects of DHA and EPA intake quartiles on LPO levels (model 1). The initial group of confounders (Model 2) consisted of socio-demographic variables, lifestyle factors and BMI. The second group of confounders (Model 3) retained all of the variables in model 2 and added energy intake, lipid prole and APOE genotype variables. The possible confounders were included as covariates in the GLM, and Bonferroni adjustment was used for all pairwise comparisons. All of statistical test were performed using the SPSS version 17.0 software (SPSS, Chicago, IL, USA), with signicance dened as Pb.05.

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3. Results 3.1. Baseline characteristics of the MCI and control groups The total study population consisted of 201 elderly individuals; their mean age was 65.24.7 years, and 77.6% of them were Malays. As has been reported elsewhere [18], 21.1% of the participants had MCI and 61.2% of those participants were women. Tables 1a and 1b depict the characteristics of the MCI (n=67) and control groups (n= 134). The mean BMI was signicantly greater in the MCI group (27.54.3kg/m2) than in the control group (26.04.8kg/m2) (Pb.05). Due to the higher BMIs in the MCI group, the daily energy intake was also expected to be higher, but the 46% intergroup difference did not reach statistical signicance. The MCI subjects performed poorer than the controls in the digit span (Pb.01), DRS (Pb.05), RAVLT total learning (Pb.05) and RAVLT delayed recall (Pb.05) subtests (Table 1a). No signicant difference was found between the two groups in either the MMSE or CDT tests. The biochemical analyses showed higher LPO levels in the MCI subjects (198.476.2 M) than in the controls (172.737.2 M) (Pb.05) (Table 1b). 3.2. Relationship between long chain omega-3 PUFAs, LPO level and cognitive performance Long chain omega-3 PUFA intake was positively correlated with the digit span (r=0.404; Pb.0001) and RAVLT delayed recall (r=0.460; Pb.0001) tests (Table 2). In addition, the plasma LPO concentrations were inversely correlated with the MMSE (r=0.180; Pb.05), digit span (r=0.171; Pb.05), RAVLT total learning (r=0.202; Pb.01) and RAVLT delayed recall (r=0.154; Pb.05) test scores. 3.3. Relationship between various variables and LPO level among the MCI and control groups As shown in Table 3, the linear regression analysis indicated a signicant inverse relationship between dietary DHA (R2=0.481; =

Table 1b The nutritional and biochemical characteristics of the MCI and control groups Characteristic MCI (n=67) N (%) APOE genotyping a 4 carrier 4 non-carrier Nutritional status b BMI (kg/m2) Energy intake (kcal/d) EPA intake (mg/d) DHA intake (mg/d) Lipid prole b TC (mmol/L) Tg (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) LPO (M) b
a b

Control (n=134) N (%) 117 (87.3) 17 (12.7) 26.04.8 1350.8250.4 89.155.4 221.5137.6 5.81.1 1.40.8 1.40.3 3.70.9 172.737.2

58 (86.6) 9 (13.4) 27.54.3 1410.8272.9 82.653.5 224.7144.7 6.01.0 1.50.8 1.40.4 3.90.9 198.476.2

.882

.025 .138 .424 .878 .178 .497 .996 .164 .013

Data are presented as number of cases (percentage). Data are presented as meanSD. Pb.05.

0.694; Pb.0001) and EPA (R2=0.431; =0.656; Pb.0001) intakes and LPO level among the MCI subjects. Other possible covariates, such as age, gender, ethnicity, exercise practice, smoking status, BMI, energy intake, lipid proles and APOE genotypes were not signicantly associated with lipid peroxidation. In addition, none of the variables were found to be signicantly correlated with circulating LPO levels in the control group.

3.4. Relationship between dietary DHA and EPA quartiles and LPO levels among the MCI subjects As a signicant relationship was found between dietary PUFA intake and LPO levels, we examine the association between LPO levels and dietary DHA and EPA quartiles. As shown in Table 4, the LPO levels among the MCI subjects decreased signicantly in the higher DHA and EPA intake quartiles (F=16.017; Pb.0001 for DHA; F=16.169; Pb.0001 for EPA). This result indicates that elderly MCI patients with lower DHA and EPA intakes had greater degrees of lipid peroxidation. When the model was adjusted for age, gender, ethnicity, exercise habits, smoking status and BMI, the associations remained signicant (Pb.0001 for DHA and EPA). The statistical signicance was not altered after the multivariate adjustment for the covariates that may affect LPO levels in human, such as energy intake, lipid proles, and APOE genotypes (model 3) (F=15.143; Pb.0001 for DHA; F=14.347; Pb.0001 for EPA). For all of the models (Models 1, 2 and 3), dietary DHA and EPA intake exerted an inuence on LPO, with

Table 1a The demographic and neuropsychological characteristics of the MCI and control groups Characteristic MCI (n= 67) n (%) Ethnicity a Malays Chinese Gender a Men Women Exercise a Yes No Smoking habit a Yes No Age b Years of education b Cognitive test (score) b MMSE Digit span DRS CDT RAVLT total learning RAVLT delayed recall
a b

Control (n= 134) n (%) 104 (77.6) 30 (22.4) 52 (38.8) 82 (61.2) 50 (37.3) 84 (62.7) 20 (14.9) 114 (85.1) 65.44.7 5.73.3 24.14.3 8.02.6 123.810.2 6.02.5 30.110.0 4.33.5

52 (77.6) 15 (22.4) 26 (38.8) 41(61.2) 22 (32.8) 45 (67.2) 12 (17.9) 55 (82.1) 64.84.6 6.33.2 23.93.1 6.91.9 120.59.0 6.22.0 26.67.8 3.22.8

1.000

1.000

.533

Table 2 The correlations among long chain omega-3 PUFA intake, LPO levels and cognitive test results Mean ( SD) Correlations with Long chain Omega-3 PUFAs r MMSE Digit span DRS CDT RAVLT total learning RAVLT delayed recall Pb.05. Pb.01. Pb.0001. 23.74.1 8.22.6 122.79.9 6.42.3 28.69.6 5.14.1 0.081 0.404 0.069 0.098 0.045 0.460 P .255 b.0001 .328 .168 .522 b.0001 LPO level r 0.180 0.171 0.072 0.015 0.202 0.154 P .012 .017 .314 .836 .005 .031

.586 .411 .231 .684 .001 .029 .497 .013 .024

Data are presented as number of cases (percentage). Data are presented as meanSD. Pb.05. Pb.01.

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Table 3 The relationships between various variables and LPO level for the MCI and control groups Factor MCI (n= 67) F statistic Age Gender Race Exercise practice Smoking habits BMI Total cholesterol Triglyceride HDL cholesterol LDL cholesterol EPA intake DHA intake Long chain omega-3 PUFAs Daily energy intake APOE4 genotyping 1.813 3.266 1.632 0.356 3.083 0.130 0.032 0.762 0.070 0.155 46.133 56.529 57.640 2.661 2.228 R2 0.029 0.051 0.026 0.006 0.048 0.002 0.001 0.012 0.001 0.003 0.431 0.481 0.486 0.045 0.035 0.170 0.225 0.161 0.076 0.219 0.046 0.023 0.111 0.034 0.050 0.656 0.694 0.697 0.213 0.188 P .183 .076 .206 .553 .084 .719 .859 .386 .792 .695 b.0001 b.0001 b.0001 .108 .141 Control (n= 134) F statistic 0.305 0.594 1.705 3.256 1.696 2.653 3.272 1.809 0.132 1.783 1.212 0.212 0.432 0.999 0.042 R2 0.002 0.005 0.014 0.024 0.013 0.020 0.025 0.022 0.001 0.014 0.009 0.002 0.003 0.008 0.000 0.048 0.067 0.243 0.156 0.113 0.141 0.157 0.063 0.032 0.116 0.096 0.040 0.058 0.089 0.018 P .582 .442 .191 .073 .195 .106 .073 .179 .717 .184 .273 .646 .512 .320 .838

Pb.0001 using linear regression analysis.

the highest lipid peroxidation concentration being observed in the rst DHA and EPA quartile.

4. Discussion The study was designed to investigate the role of dietary long chain omega-3 PUFAs in modulating the degree of lipid peroxidation among elderly MCI patients. LPO was chosen as the biomarker for lipid peroxidation primarily due to the shortcomings of the commonly used indirect colorimetric assay measurements: (i) HNE is formed from -6 PUFA hydroperoxides and can be catalysed by transition metal ions such as ferrous ions, which leads to an underestimation of lipid peroxidation; (ii) MDA is also produced by the platelet enzyme thromboxane synthase during whole blood clotting and platelet activation, which can lead to a gross over-estimation of lipid peroxidation; and(iii) potential confounders, such as smoking and diabetes, may increase the levels of thiobarbituric acid reactive substances (TBARS), MDA and HNE [27]. Omega-3 PUFA intake was found to be positively correlated with the cognitive function, particularly attention, short term memory and recall capabilities. Meanwhile, LPO concentrations were inversely associated with global cognitive function, attention, short-term memory and immediate and delayed learning. The ndings are consistent with the previous studies, in which increasing plasma omega-3 PUFA concentrations signicantly reduced cognitive decline [28] and in which higher plasma phosphatidylcholine DHA [29] and

omega-3 PUFA[30] concentrations reduced the risk of dementia and AD in late life. In addition, the LPO levels were higher in the MCI group than in the control group, which is consistent with previous studies [3,31]. Autopsy studies have also found increased TBARS, MDA and HNE in the brains of MCI patients [3235], indicating that lipid peroxidation is a potential biomarker for identifying those MCI subjects who are at greater risk for AD. Nevertheless, conicting opinions on using biomarkers to demonstrate oxidative stress in neurodegenerative diseases have been expressed. This disagreement has been attributed to the blood brain barrier being intact in most neurodegenerative disease, so that oxidative stress markers are not reected in systemic circulation [32]. An important nding of this study was the effect of DHA and EPA intake on reducing LPO levels among the elderly MCI patients. Similarly, Richard et al. [11] have presented cellular evidence suggesting that omega-3 PUFAs may act as an indirect antioxidant rather than a pro-oxidant in vascular endothelial cells with lower ROS production. Mazire et al. [14] have also indicated that omega-3 PUFAs are more effective than omega-6 PUFAs for decreasing TBARS, LDL peroxidation and superoxide anion secretion in human endothelial cells. Su [36] has also suggested that the anti-oxidative stress induced by omega-3 PUFAs protects neuron in the aged, damaged, and AD brain. Meanwhile, rats and Tg2576 transgenic mice fed DHAenriched diets show a similar reductions in cerebral lipid peroxide and oxidised proteins level [37,38]. However, the LPO levels in the control group were not inuenced by omega-3 PUFA intake. This result may be related to better

Table 4 The mean LPO levels in the MCI group by dietary DHA and EPA quartiles F statistic LPO level (M) 1st DHA quartile (070.11 mg/d) (n= 15) 273.4 272.0 287.8 262.1 258.8 271.4 (243.4303.4) a (241.3302.6) a (253.9321.7) a (233.2291.0)a (228.5289.1)a (235.6307.2)a 2nd DHA quartile (70.12202.32 mg/d) (n= 18) 214.1 212.1 210.6 227.1 227.7 228.3 (186.7241.4)bc (183.5240.7)bc (182.1239.0)b (199.9254.4)a (199.0256.4)a (200.0256.6)a 3rd DHA quartile (202.33354.52 mg/d) (n= 14) 167.2 168.6 159.6 150.2 152.7 147.3 (136.2198.2)bc,d (137.0200.1)bc,d (127.8191.4)b (122.1178.3)b,c (124.1181.4)b,c (119.1175.5)b,c 4th DHA quartile ( 354.53 mg/d) (n= 16) 137.6 140.0 146.7 138.5 138.4 145.2 (108.6166.6)bc,d (109.8170.1)bc,d (116.1177.3)b (105.1171.9)b,c (103.6173.3)b,c (108.3182.2)b,c b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 P

Model Model Model Model Model Model

1 2 3 1 2 3

16.017 14.099 15.143 16.169 14.222 14.347

Model 1: no adjustment. Model 2: adjusted for age, gender, ethnicity, exercise habit, smoking status and BMI. Model 3: adjusted as model 2 and additionally adjusted for energy intake, lipid proles, and APOE genotypes. Data are presented as mean (95% CI) using Generalized Linear Model. Values in the same row that do not share the same superscript letter are signicantly different. Pb.0001.

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antioxidant defences in the control group than in the MCI patients [3]. Although the only statistically signicant association found was between LPO levels and DHA and EPA intake, there were a number of associations that almost reached statistical signicance (gender and smoking habits for the MCI group and exercise and total cholesterol for the control group), suggesting that these well known modulators of oxidative stress may be potential risk factors for elevated lipid peroxidation. The mechanisms by which omega-3 PUFAs achieve their protective effect has not been well established. A proposed mechanism has suggested that DHA is capable of accelerating the production of endogenous antioxidant enzymes [39], which play a prominent role in activating the antioxidant defence system and which enhance the cerebral activities of catalyse, glutathione peroxidase and glutathione [40]. Additionally, DHA is important for regulating neuronal transmission by maintaining the structural integrity of neuronal membranes, attenuating amyloid activity, reducing inammation and cholesterol, and inhibiting the secretion of pro-inammatory cytokines [41]. The mechanisms by which DHA, EPA and gene expression reduce lipid peroxidation among MCI patients need further investigation. This is an early study investigating the impact of omega-3 PUFA consumption on lipid peroxidation status among the elderly MCI patients, and it bridges a gap in the current research literature. Several covariates were considered, which eliminates the potential confounding effects of known associations between oxidative stress and these covariates. Direct measurements of the peripheral hydroperoxide concentrations yielded better estimates lipid peroxidation status. However, it would be more valuable to have peripheral composition of omega-3 PUFAs to better interpret the correlation with lipid peroxidation. FFQ was used to capture omega-3 PUFA intake, and the accuracy of the reported data depended on the subject's ability to recall the specic foods consumed. Discrepancies that have been observed between observational and interventional studies suggest that the effects of diet on cognition can only be observed after long-term treatment. However, the dietary method used to estimate DHA and EPA intake in the present work was valid, even for larger scale studies [42]. Consistent with results reported for the United States [43], over 80% of the study participants were APOE 4 carriers. However, a local study has reported that 3/3 was the most prevalent APOE genotype among Malays [44]. Given these limitations, additional research to determine the APOE genetic polymorphisms in a larger random sample of elderly Malaysians is recommended. In addition, it would be useful to quantify the extent to which quantication of endogenous oxidative defence systems, such as superoxide dismutase and glutathione peroxidase, modulate lipid peroxidation. This study demonstrated that greater lipid peroxidation occurs among MCI patients, and it is possible that oxidative imbalances precede cognitive deterioration in elderly individuals. Higher DHA and EPA intakes reduce LPO levels among the elderly MCI patients. These observations, although not widely explored, are promising for the importance of EPA and DHA metabolism in modulating lipid peroxidation among cognitively impaired elderly patients. Future investigations should provide a better understanding of the clinical signicance of EPA and DHA levels, especially during the incorporative phase, and genetic susceptibility in elderly MCI patients.

References
[1] Winblad B, Palmer K, Kivipelto M, Jelic V, Fratiglioni L, Wahlund LO, et al. Mild cognitive impairment-beyond controversies, towards a consensus: report of the International Working Group on Mild Cognitive Impairment. J Intern Med 2004;3: 2406. [2] Baldeiras I, Santana I, Proenc MT, Garrucho MH, Pascoal R, Rodrigues A, et al. Peripheral oxidative damage in mild cognitive impairment and mild Alzheimer's disease. J Alzheimer's Dis 2008;15:11728. [3] Padurariu M, Ciobicab A, Hritcu L, Stoicaa B, Bilda W, Stefanescu C. Changes of some oxidative stress markers in the serum of patients with mild cognitive impairment and Alzheimer's disease. Neurosci Lett 2010;469:610. [4] Butterfield DA, Castegna A, Pocernich CB, Drake J, Scapagnini G, Calabrese V. Nutritional approaches to combat oxidative stress in Alzheimer's disease. J Nutr Biochem 2002;13:44461. [5] Markesbery WR, Lovell MA. Damage to lipids, proteins, DNA, and RNA in mild cognitive impairment. Arch Neurol 2007;7:9546. [6] Mangialasche F, Cristina Polidori M, Monastero R, Ercolani S, Camarda C, Cecchetti R, et al. Biomarkers of oxidative and nitrosative damage in Alzheimer's disease and mild cognitive impairment. Ageing Research Rev 2009;8:285305. [7] Carini M, Aldini G, Facino RM. Mass spectrometry for detection of 4-hydroxytrans-2-nonenal (HNE) adducts with peptides and proteins. Mass Spectrom Rev 2004;4:2815. [8] Yehuda ES, Rabinovitz S, Carasso RL, Mostofsky DI. The role of polyunsaturated fatty acids in restoring the aging neuronal membrane. Neurobiol Aging 2002;5: 84353. [9] Tai CC, Ding ST. N-3 polyunsaturated fatty acids regulate lipid metabolism through several inflammation mediators: mechanisms and implications for obesity prevention. J Nutr Biochem 2010;21:35763. [10] Lin YH, Shah S, Salem N. Altered essential fatty acid metabolism and composition in rat liver, plasma, heart and brain after microalgal DHA addition to the diet. J Nutr Biochem 2011;22:75865. [11] Richard D, Kefi K, Barbe U, Bausero P, Visioli F. Polyunsaturated fatty acids as antioxidants. Pharmacol Res 2008;57:4515. [12] Kameda K, Matsunaga T, Abe N, Hanada H, Ishizaka H, Ono H, et al. Correlation of oxidative stress with activity of matrix metalloproteinase in patients with coronary artery disease: Possible role for left ventricular remodelling. Eur Heart J 2003;24:21805. [13] Wright SA, O'Prey FM, McHenry MT, Leahey WJ, Devine AB, Duffy EM, et al. A randomised interventional trial of omega-3 polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus. Ann Rheum Dis 2008;67:8418. [14] Mazire C, Dantin F, Conte MA, Deqonville J, Ali D, Dubois F, et al. Polyunsaturated fatty acid enrichment enhances endothelial cell-induced low-density-lipoprotein peroxidation. Biochem J 1998;336:5762. [15] Roberts RO, Cerhan JR, Geda YE, Knopman DS, Cha RH, Christianson TJH, et al. Polyunsaturated fatty acids and reduced odds of MCI: the Mayo Clinic Study of Aging. JAD 2010;21:85365. [16] Solfrizzi V, Frisardi V, Capurso C, D'Introno A, Colacicco AM, Vendemiale G, et al. Dietary fatty acids in dementia and predementia syndromes: epidemiological evidence and possible underlying mechanisms. Ageing Res Rev 2010;9:18499. [17] Petersen RC. Mild cognitive impairment as a diagnostic entity. J Intern Med 2004;256:18394. [18] Lee LK, Shahar S, Chin AV, Mohammad Yusoff NA, Rajab NF, Aziz S. Prevalence of gender disparities and predictors affecting the occurrence of mild cognitive impairment (MCI). Arch Gerontol Geriatr 2012;54:18591. [19] Ibrahim NM, Shohaimi S, Chong HT, Abdul Rahman AH, Razali R, Esther E, et al. Validation study of the Mini-Mental State Examination in a Malay-Speaking elderly population in Malaysia. Dement Geriatr Cogn Disord 2009;27:24753. [20] Mattis S. Dementia rating scale: professional manual. Odessa (Fla): Psychological Assessment Resources Inc; 1988. [21] Rey A. L'examenclinique en psychologie. Paris: Presses Universitaires de France; 1964. [22] Wechsler D. Wechsler Adult Intelligence Scale. 3rd ed. San Antonio (Tex): The Psychological Corporation; 1997. [23] Sunderland T, Hill JL, Mellow AM, Lawlor BA, Gundersheimer J, Newhouse PA, et al. Clock drawing in Alzheimer's disease: a novel measure of dementia severity. J Am Geriatr Soc 1989;37:7259. [24] Tee ES, Ismail MN, Nasir MA, Khatijah I. Nutrient composition of Malaysian foods. 4th ed. Kuala Lumpur: Institute of Medical Research; 1997. [25] National Food Authority. Composition of Foods, Australia (COFA), volume 7. Canberra: Australian Government Publishing Services; 1995. [26] Hixson J, Vernier D. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage by HhaI. J Lipid Res 1990;31:5458. [27] Martin-Gallan P, Carrascosa A, Gussinye M, Dominguez C. Biomarkers of diabetesassociated oxidative stress and antioxidant status in young diabetic patients with or without subclinical complications. Free Radic Biol Med 2003;12:156374. [28] van de Rest O, Geleijnse JM, Kok FJ, van Staveren WA, Dullemeijer C, Olderikkert MG, et al. Effect of fish oil on cognitive performance in older subjects: a randomized, controlled trial. Neurology 2008;71:4308. [29] Schaefer EJ, Bongard V, Beiser AS, Lamon-Fava S, Robins SJ, Au R, et al. Plasma phosphatidylcholinedocosahexaenoic acid content and risk of dementia and Alzheimer disease: the Framingham Heart Study. Arch Neurol 2006;63:154550.

Acknowledgments The authors acknowledge the participants for their involvement in this study.

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[30] Boudrault C, Bazinet RP, Ma DWL. Experimental models and mechanisms underlying the protective effects of n-3 polyunsaturated fatty acids in Alzheimer's disease. J Nutr Biochem 2009;20:110. [31] Greilberger J, Koidl C, Greilberger M, Lamprecht M, Schroecksnadel K, Leblhuber F, et al. Malondialdehyde, carbonyl proteins and albumin-disulphide as useful oxidative markers in mild cognitive impairment and Alzheimer's disease. Free Radical Res 2008;42:6338. [32] Pratico D, Clark CM, Liun F, Rokach J, Lee VY, Trojanowski JQ. Increase of brain oxidative stress in mild cognitive impairment: a possible predictor of Alzheimer disease. Arch Neurol 2002;59:9726. [33] Keller JN, Schmitt FA, Scheff SW, Ding Q, Chen Q, Butterfield DA, et al. Evidence of increased oxidative damage in subjects with mild cognitive impairment. Neurology 2005;64(7):11526. [34] Butterfield DA, Reed T, Perluigi M, De Marco C, Coccia R, Cini C, et al. Elevated proteinbound levels of the lipid peroxidation product, 4-hydroxy-2-nonenal, in brain from persons with mild cognitive impairment. Neurosci Lett 2006;397:1703. [35] Williams TI, Lynn BC, Markesbery WR, Lovell MA. Increased levels of 4hydroxynonenal and acrolein, neurotoxic markers of lipid peroxidation, in the brain in mild cognitive impairment and early Alzheimer's disease. Neurobiol Aging 2006;27:10949. [36] Su HM. Mechanisms of n-3 fatty acid-mediated development and maintenance of learning memory performance. J Nutr Biochem 2010;21:36473.

[37] Hossain MS, Hashimoto M, Masumura S. Influence of docosahexaenoic acid on cerebral lipid peroxide level in aged rats with and without hypercholesterolemia. Neurosci Lett 1998;244:15760. [38] Kubo K, Saito M, Tadokoro T, Maekawa A. Dietary docosahexaenoic acid dose not promote lipid peroxidation in rat tissue to the extent expected from peroxidizability index of the lipids. Biosci Biotech Bioch 1998;62:1698706. [39] Yavin E, Brand A, Green P. Docosahexaenoic acid abundance in the brain: a biodevice to combat oxidative stress. Nutr Neurosci 2002;5:14957. [40] Hossain MS, Hashimoto M, Gamoh S, Masumura S. Antioxidative effects of docosahexaenoic acid in the cerebrum versus cerebellum and brainstem of aged hypercholesterolemic rats. J Neurochem 1999;72:11338. [41] Cunnane SC, Plourde M, Pifferi F, Fart C, Barberger-Gateau P. Fish, docosahexaenoic acid and Alzheimer's disease. Prog Lipid Res 2009;48:23956. [42] Bloomer RJ, Kabir MM, Trepanowski JF, Canale RE, Farney TM. A 21 day Daniel Fast improves selected biomarkers of antioxidant status and oxidative stress in men and women. Nutr Metab 2011;8:1725. [43] Saunders AM, Hulette C, Welsh-Bohmer KA, Schmechel DE, Crain B, Burke JR, et al. Specificity, sensitivity, and predictive value of apolipoprotein-E genotyping for sporadic Alzheimer's disease. Lancet 1996;348:903. [44] Seet WT, Tan JA, Mary A, Tan SY. Apolipoprotein E genotyping in the Malay, Chinese and Indian ethnic groups in Malaysia a study on the distribution of the different apoE allele and genotypes. Clinical Chimica Acta 2004;340:2015.