Abstract
Low tissue levels of the major marine ω3 fatty acids (FAs) DHA and EPA are found in Alzheimer’s disease (AD). We investigated if healthy proxies to AD patients have higher levels of these ω3 FAs. We observed lower levels of EPA and DHA in subcutaneous adipose tissue biopsies from 64 AD patients compared with 16 cognitively healthy proxies. No significant difference was observed when pairwise comparisons were made between a subset of 16 AD patients and their co-habiting proxies. Larger studies are needed to replicate these findings and to determine if they could depend on FA intake or differences in metabolism.
INTRODUCTION
Various trials indicate that fatty acid (FA) intake, especially of ω3 FA docosahexaenoic acid (DHA), plays a significant role in the development and maintenance of cognitive function [1]. High intakes of oily fish, rich in DHA, may prevent cognitive decline at a population level [2, 3]. Lower levels of DHA and eicosapentaenoic acid (EPA) in plasma and in the brain have been found in AD patients compared to healthy controls [4, 5–7].
Low tissue levels of DHA and EPA in subjects with AD could be due to reduced intake of marine food. However, it cannot be ruled out that the reduced levels could be related to impaired uptake or dysregulated metabolism of DHA and EPA. Tissue levels of essential FAs, e.g., DHA and EPA, usually reflect dietary intake, but quantitative data about rate of incorporation as a function of intake as well as of AD-specific changes of FA metabolism are scarce [8, 9].
Previous studies have shown significant correlations between ω3 FA intake and plasma and adipose tissue levels of EPA or DHA [1, 10]. A few randomized controlled trials (RCT) in subjects with mild cognitive impairment (MCI), in frail elderly persons, and in AD patients have reported effects on plasma [11, 12] and cerebrospinal fluid (CSF) [13]. To our knowledge, no previous study has reported adipose tissue levels of EPA and DHA in AD patients or addressed possible differences in ω3 FA levels between AD patients and their cohabitants. It may be assumed that a common household reflects common food preferences and access to similar food.
Thus, the primary aim was to study if the adipose tissue of patients with mild AD has a different major marine long-chained FA composition compared to that of healthy cohabiting proxies. A secondary aim was to see if levels of DHA and EPA in subcutaneous (sc) adipose tissue correlate with those in CSF and plasma.
MATERIALS AND METHODS
Data for this study was obtained from the OmegAD trial, a double-blind RCT in 204 patients with mild to moderate AD who were given either a DHA-enriched ω3 FA supplement (1.7 g DHA and 0.4 g EPA) or a placebo for 6 months [14]. The Supplementary Material gives details about inclusion criteria.
Fatty acids in adipose tissue in patients
At baseline, biopsies from upper arm sc adipose tissue were obtained from two subgroups of patients: the first (n = 40) and the last (n = 24) group of patients included in the OmegAD trial.
All tissue, plasma and CSF samples were stored in an identical way at –80C before FA profiles in plasma and sc adipose tissue were determined by gas chromatography [14, 15]. The analyzes were performed in total FAs after hydrolysis of esters and the method is thoroughly described by Boberg et al. [15]. Plasma levels of DHA and EPA were obtained from all 64 patients, whereas DHA and EPA in CSF were only analyzed in the first subgroup of patients. The full report on CSF FA data is given in [13]. Each FA was expressed as a percentage of the total FAs in plasma and adipose tissue, and primarily as concentration (ng/ml) in CSF. For comparisons between plasma and CSF FA values, the latter were converted from ng per ml to proportion (%) of total FA weight.
Examinations of healthy cohabitants
Biopsies from sc adipose tissue were also obtained from 16 healthy co-habitants of the last group of patients included, i.e., persons that shared the same household as the patients with AD. None of the co-habitants were “blood relatives”. The majority of them were spouses of the participants of the OmegAD Trial. The cohabitants underwent cognitive screening with the Mini-Mental State Examination (MMSE) and were asked about heredity, i.e., relatives with dementia or AD (Supplementary Table 1).
Statistical analyses
Data is presented as mean±standard deviation (SD). The Shapiro-Wilk test was used to examine the normality of variables. Depending on the distribution of the variables, independent samples t-test or Mann Whitney U-test were used to analyze variations between the groups. Pearson’s or Spearman’s correlation coefficients were calculated to measure the linear correlation between variables. Regression analysis was used to calculate the adjusted p-value where AD patients, their cohabiting relatives, gender, and age were included as independent variables in the models. The IBM SPSS Statistics software and Statistica® 9.1 software package were used for the statistical calculations.
Ethical considerations
Good Clinical Practice Guidelines and ethical principles of the Declaration of Helsinki were applied to the study. Both patient and caregiver gave written informed consent prior to study entry. The Local Ethics Committee at Karolinska University Hospital Huddinge approved the study (Dnr 29100/2001, 2010/1434-32).
RESULTS
In total, levels of FAs in adipose tissue from 64 patients (55% males; 73±8 years, 50 (78%) were APOE ɛ4 carriers) and 16 cohabitants (69% males; 77±7 years) were analyzed. Basic characteristics are displayed in Supplementary Table 1. The cohabitants were more often male, slightly older than the patients, and almost 20% of them reported heredity for dementia or AD.
EPA and DHA in adipose tissue
The levels of EPA and DHA were significantly lower in the total sample of AD-patients (n = 64) versus the proxy controls (n = 16; p < 0.001 and p = 0.008, respectively; Table 1). The data was run with and without log transformation and results were similar. A similar pattern appeared when comparisons were made in the sub-sample of 16 pair-matched couples (patient and cohabiting proxy). A difference in levels of EPA between patients and proxies was noted (p = 0.062); however, this did not reach statistical significance, likely due to the small number of subjects. The levels of EPA and DHA did not differ between patients with and without proxies or between patients with or without APOE ɛ4 carrier status (data not shown). Gender differences in the pair-matched couples were not meaningful to analyze because of an insufficient number of female relatives.
The relative proportion (%) of fatty acids in adipose tissue in AD-patients and their cohabiting relatives
Data is shown as mean±standard deviation and median (Q1-Q3; interquartile range). Adjusted p-values are calculated by regression models where AD-patients/their cohabiting relatives, gender, and age are included as independent variables in the models. MCalculated by Mann-Whitney U test. TCalculated by independent samples t-test. FA profiles in serum and sc adipose tissue were determined by gas chromatography [14, 15]. Each FA was expressed as percentage of total FAs.
Comparisons between EPA and DHA levels in adipose tissue, plasma, and CSF
Significant correlations were found between plasma and adipose tissue levels of EPA (rho = 0.44, p < 0.001) and DHA (rho = 0.49, p < 0.001) (Table 2). Significant correlations were also found between CSF (n = 34) and adipose tissue levels of EPA (rho = 0.46, p = 0.006). Associations of tissue FAs with plasma FAs and CSF FAs were also confirmed by regression analyses where age, gender and weight were included as independent variables in the model (Table 2).
Associations of fatty acids (in proportion of total fatty acids) in adipose tissue with plasma fatty acids and CSF fatty acids in AD patients
Adjusted p-values are calculated by regression models where age, gender and weight are included as independent variables in the models. PPearson’s correlation coefficient. SSpearman’s correlation coefficient.
DISCUSSION
This study indicates that the patients with mild AD (n = 64) had lower levels of the ω3 FAs EPA and DHA in sc adipose tissue compared to the healthy proxies (n = 16). A recent systematic review and meta-analysis of plasma nutrient status in AD patients confirms that patients with AD have lower plasma levels of DHA compared to healthy control subjects [16]. Whether reduced tissue levels of EPA and DHA precede AD or whether this is an effect of the progressive cognitive decline during the AD trajectory is not yet clear. EPA and DHA are considered to be essential FAs for nervous tissue, but the intake of alpha-linolenic acid (ALA; 18 : 3ω3) may contribute to small amounts of EPA and DHA after elongation and desaturation [17]. Thus, reduced tissue levels of EPA and DHA may reflect a reduced marine food intake. Interestingly, ALA levels in adipose tissue did not differ between the patients and their cohabitants. It might be speculated that the differences in adipose DHA and EPA concentrations may not be related to reduced dietary intake but perhaps to disturbed catabolism, uptake, or altered ω3 FA metabolism in patients with AD [18]. It is therefore of importance to compare ω3 FA status in patients with AD with their cohabiting relatives, given the presumption that their food intake is similar. Significant correlations were found in the patients between plasma (n = 64) and adipose tissue levels of EPA and DHA and between CSF (n = 34) and adipose tissue levels of EPA. However, there was no association between CSF DHA and adipose tissue DHA, which may be interpreted as a limitation of sample size.
In this study, we compared the levels of DHA and EPA in sc adipose tissue in AD patients with those in healthy proxies, of which 15 out of 16 couples were cohabitants and shared meals together. A novel finding was that the levels of DHA and EPA were lower in the 64 AD patients when compared to 16 healthy cohabitants. However, this difference was no longer statistically significant when only the 16 pair-matched patients and cohabitants were compared, although the absolute differences were similar. Thus, the low number of proxies in this subset of individuals could explain the non-significant outcome.
The differences seen in plasma, CSF, and adipose tissue ω3 FAs between AD patients and healthy subjects have also been reported for several other nutrients such as B-vitamins, vitamin C, vitamin E, and some micronutrients [16]. These findings indicate the possibility of impaired availability of nutrients for the brain [19]. Lower endogenous biosynthesis of DHA in the liver has been suggested as one metabolic pathway that is altered in AD [18]. These nutrient deficiencies often occur early in AD, long before weight loss and malnutrition ensue [20]. Patients with AD may benefit from a higher nutrient intake to address the disease-specific requirements and to compensate for lower nutrient availability. More research is warranted to investigate the pathophysiology causing the reduced nutrient concentrations in AD.
Plasma non-esterified FAs (NEFAs) that are mobilized from adipose tissue have been suggested as a surrogate marker in adipose tissue. Since biopsy of adipose tissue is an invasive procedure, alternative methods of investigation may be preferred. In an intervention trial where healthy middle-aged subjects were supplemented with EPA and DHA, associations were found between adipose tissue EPA and DHA, and corresponding concentrations in plasma non-esterified FAs, however no association was found after the intervention. Plasma NEFAs may therefore not be a suitable surrogate for ω3 FAs in adipose tissue [21].
Conclusions
This study indicates that patients with mild AD have lower levels of the ω3 FAs EPA and DHA in sc adipose tissue compared to healthy proxies, implying that either the AD patients intake of ω3 FAs from food is reduced or their metabolism of the corresponding FAs is altered. Larger study samples, where patients and their healthy proxies are investigated simultaneously and where sc tissue levels of ω3 FAs data are correlated to data on fish and seafood intake, should be performed to give more conclusive results regarding whether the potential variations in tissue ω3 FA levels depend on reduced intake or increased turnover.
Footnotes
ACKNOWLEDGMENTS
Financial support was provided through The Regional Agreement on Medical Training and Clinical Research (ALF) between Stockholm County Council and Karolinska Institutet, Medical Research Council, Funds of Capio, Demensförbundet, Gamla Tjänarinnor, Swedish Alzheimer Foundation, Odd Fellow, Swedish Nutrition Foundation, Gun och Bertil Stohnes Stiftelse, Swedish Society of Physicians and Lion’s Sweden.
