Here, we describe that 6 months of dietary supplementation of a DHA rich fish oil formulation to elderly humans afflicted with AD conferred significant up- or down-regulation of several genes. They represent a wide variety of cellular functions. Notably, many of these are associated with inflammatory reactions (viz
), others with neuroinflammatory disorders (viz
), two processes which are highly relevant for actions of n-3 FAs and the aim of the OmegAD study to investigate. Some of the genes were found in both categories, emphasizing the inflammatory component of AD process 
. Moreover, only 1 (of 19) genes was significantly changed in the placebo treated group. Finally, the statistically significant relation between changes of plasma DHA and EPA levels and of the RHOB
genes is intriguing.
The four previously mentioned genome wide expression studies in baboons, rodents and the one in healthy subjects 
noted changes in 5–1000 genes in brains, livers and PBMC, respectively, after dietary DHA or EPA rich fish oil interventions for 3–10 weeks in rodents and 26 weeks in humans. However and surprisingly, none of those genes coincided with what we describe here as significantly up- or down-regulated. A possible explanation of these inconsistencies in results might be related to species, human populations and ages, FA types, doses, duration as well as to target organs. Thus, our study presents unique data on genes of relevance to long-term dietary supplementation with a DHA-rich preparation to aged and AD-afflicted humans.
Array techniques with a restricted set of gene probes (app. 3000 genes or less) were used in a few n-3 FA animal studies. In one, on brain tissue, the fish oil group displayed almost the same expression profile as the control group 
. Another study found an up-regulation of the transthyretin gene in the hippocampus 
, whereas we did not in our study on human PBMC. In a third murine study, genes encoding for IL-1α, IL-1β or NO synthase were unaltered 
, as in our study. Two months of a DHA rich fish oil supplementation modified 77 of 588 studied genes in human lymphocytes 
but, again, none was genes we identified as up-or down-regulated.
The recently published study on healthy humans given an EPA rich fish oil 
(with total n-3 FA doses close to ours and for a similar time) observed changes in more than 1000 genes, where the magnitude of changes was often very small. The reason for the outfall in terms of number of genes can probably be related to using an array covering 17 000 genes, including approximately twice as many subjects as in our study, and using a nominal p-value 0.05 as threshold to determine significantly altered gene expression on microarray data. The latter makes the results difficult to compare with our analysis of microarray data with SAM, which takes multiple comparisons into account. Since only a fraction of all changed genes were listed in 
, we do not know if changes were seen in the same genes as in our study. Of note, out of 99 independent transcripts (changed by EPA) that could be identified in 
, 16 transcripts were not represented with probesets on our Focus array. Another 19 transcripts were detected in less than 29 of 32 arrays, and displayed very low signals; it has previously been demonstrated that variability in gene expression is a function of absolute expression, i.e. a lowly expressed gene is more variable than a highly expressed gene 
. Results for such genes have an increased risk of being false positive and are also more difficult to confirm in independent datasets. In addition, some of the remaining 54 EPA-regulated transcripts in 
gave low signals on our Focus array. Together, these factors can partially explain why we could not confirm the findings by Bouwens et al 
Several studies have examined the effects of fish oils on individual human genes and corresponding protein production, documenting, inter alia, diminished ex vivo pro-inflammatory cytokine production in mononuclear blood cells, e.g. TNF-α and IL-1β 
and IL-6 
and decreased TNF-α and IL-6 in a dose-dependent manner 
. Fish oil also lowered gene expression of TNF-α in renal allograft after 3 months of treatment 
. These findings could, however, not consistently be repeated in other studies 
. Again, the reason for these discrepancies may relate to n-3 FA dosage, treatment time, whether DHA or EPA was the predominant FA (since they may regulate genes differently) 
, the cell type and the experimental design. This is further illustrated by rather heterogeneous results from animal studies 
where species differences and age effects also are of significance.
As shown here, EPA as well as DHA plasma levels was enhanced in a similar way, suggesting that DHA was converted to EPA to some extent or that DHA was specifically cleared from plasma. This makes it difficult to attribute gene changes to one FA. However, DHA enriched formulas were rarely used in previous in vivo studies.
In a previous publication from the OmegAD study 
, we reported about changes of pro-inflammatory cytokine and growth factor production from LPS stimulated blood mononuclear cells ex vivo. However, we are unable to show any changes in corresponding genes. This might be due to our gene expression data is based on quiescent PBMCs, without LPS activation.
and after dietary supplementation, DHA increased the sortilin-1 receptor (SORL1
) mRNA and protein in murine cortical neurons 
, but decreases have also been observed 
regulates the amyloid precursor protein and this protein is also elevated in aged DHA depleted mice 
. In our study we found that n-3 FA down-regulated the expression of the SORL1
gene which is in agreement with Perez et al 
Our DHA enriched n-3 FAs supplementation for 6 months caused effects with higher magnitudes on several genes compared with placebo treatment. Moreover, the genes RHOB and ANAPC5 also showed a statistically significant correlation with the changes of both plasma DHA and EPA levels, making them interesting candidates for further studies on effects of n-3 FAs.
The magnitude of the gene expression changes observed here might appear rather small, since they ranged between +72 and – 30 per cent. This is, however, what might be expected in dietary supplementation studies in animals and humans 
. In contrast, in vitro
studies or in short-term animal studies larger changes might be found. One can anticipate that acute and large effects will vane with prolonged exposure because of an adaptation over time.
The present study gives novel information on mechanisms for marine lipids, suggesting that dietary n-3 FA supplementation affected expression of genes that might influence inflammatory processes and could be of significance for Alzheimer's disease. Recently, the significance of n-3 FA blood levels for telomeric aging in patients with coronary heart disease was demonstrated