Act that healthy humans were supplemented with n? PUFA in this study, as opposed to the 15900046 rodent studies in which a group of animals were developmentally deprived of n? PUFA and compared to controls. Thus, the possibility of dietary depletion of n? PUFA leading to a reduction in Epigenetics striatal VMAT2 availability in humans cannot be excluded based on 
the six-month supplementation data. Because individuals with diets deficient in n? PUFA are likely to have less RBC DHA/EPA, we evaluated whether lower RBC Table 1. RBC fatty acid composition analysis.DHA/EPA levels are associated with lower striatal VMAT2 availability in subjects before supplementation. Contrary to this hypothesis, we found no relationship between the RBC DHA/ EPA levels and striatal [11C]DTBZ BPND. Taken together these data do not support an effect for n? PUFA on striatal VMAT2 in healthy adults. Two interesting observations are reported in this study. The first is that in this group of young adults superior working memory performance in the inhibitor 3-back condition prior to supplementation was correlated with higher RBC DHA. This finding is consistent with a previous report in which higher serum DHA was related to superior performance on tests of non verbal reasoning and working memory in a relatively large cohort of middle aged adults [2]. Second, there was an improvement in working memory performance in the 3-back condition after six months of n? PUFA supplementation. Although, practice-effects cannot be ruled out as the reason for this observation in this cohort, this result is consistent with some clinical trials suggesting that n? PUFA (fish oil) supplementation improves cognitive functioning in elderly adults with mild to no cognitive impairment [33?7]. Surprisingly, 3-back performance improvement was significant despite the fact that there was no correlation between changes in AHR and RBC DHA/EPA levels following supplementation with n? PUFA. But, when individuals were stratified into two groups based on their pre-supplementation DHA levels (i.e., less than or greater than 3 mol of total fatty acid pool) we found that the mean change in AHR 3-back was 0.2960.18 in the low DHA group (n = 6 Table 2. Adjusted hit rate from the n-back working memory task.Type n? PUFAPUFA ALA DHA EPAPre- n3 PUFA 0.460.1 2.961.0 0.460.1 21.765.1 13.962.Post- n3 PUFA 0.460.1 5.161.2 1.860.8 21.963.8 12.262.t 0.df p-value 10 0.92 n-back 1-back 2-back 3-back Pre- n3 PUFA 0.9860.04 0.9360.10 0.6560.27 Post- n3 PUFA 0.9960.02 0.9460.09 0.8060.29.89 10 ,0.01 26.30 10 ,0.01 20.13 10 0.90 3.49 10 0.tdfp-value 0.17 0.70 0.21.480 10 20.399 10 22.292n? PUFALA AAValues are mean and standard deviation (SD), n = 11 per condition. p-values are from two-tailed, paired t tests; t is t statistic; df is degrees of freedom. doi:10.1371/journal.pone.0046832.tValues are mean and standard deviation (SD), n = 11 per condition. p-values are from two-tailed, paired t tests; t is t statistic; df is degrees of freedom. doi:10.1371/journal.pone.0046832.tOmega-3 Fatty Acid Supplementation and VMATFigure 2. Shows the relationship between pre-supplementation RBC DHA or EPA in x-axis and pre-supplementation performance (AHR) in 3-back test in y-axis. The AHR ranges from 1 (best performance) to 21 (worst performance), with a score of 0 corresponding to performance at chance level. RBC DHA (Panel A), but not EPA (Panel B) was associated with performance in the task. doi:10.1371/journal.pone.0046832.gsubjects) and 20.0160.14 in the high.Act that healthy humans were supplemented with n? PUFA in this study, as opposed to the 15900046 rodent studies in which a group of animals were developmentally deprived of n? PUFA and compared to controls. Thus, the possibility of dietary depletion of n? PUFA leading to a reduction in striatal VMAT2 availability in humans cannot be excluded based on the six-month supplementation data. Because individuals with diets deficient in n? PUFA are likely to have less RBC DHA/EPA, we evaluated whether lower RBC Table 1. RBC fatty acid composition analysis.DHA/EPA levels are associated with lower striatal VMAT2 availability in subjects before supplementation. Contrary to this hypothesis, we found no relationship between the RBC DHA/ EPA levels and striatal [11C]DTBZ BPND. Taken together these data do not support an effect for n? PUFA on striatal VMAT2 in healthy adults. Two interesting observations are reported in this study. The first is that in this group of young adults superior working memory performance in the 3-back condition prior 
to supplementation was correlated with higher RBC DHA. This finding is consistent with a previous report in which higher serum DHA was related to superior performance on tests of non verbal reasoning and working memory in a relatively large cohort of middle aged adults [2]. Second, there was an improvement in working memory performance in the 3-back condition after six months of n? PUFA supplementation. Although, practice-effects cannot be ruled out as the reason for this observation in this cohort, this result is consistent with some clinical trials suggesting that n? PUFA (fish oil) supplementation improves cognitive functioning in elderly adults with mild to no cognitive impairment [33?7]. Surprisingly, 3-back performance improvement was significant despite the fact that there was no correlation between changes in AHR and RBC DHA/EPA levels following supplementation with n? PUFA. But, when individuals were stratified into two groups based on their pre-supplementation DHA levels (i.e., less than or greater than 3 mol of total fatty acid pool) we found that the mean change in AHR 3-back was 0.2960.18 in the low DHA group (n = 6 Table 2. Adjusted hit rate from the n-back working memory task.Type n? PUFAPUFA ALA DHA EPAPre- n3 PUFA 0.460.1 2.961.0 0.460.1 21.765.1 13.962.Post- n3 PUFA 0.460.1 5.161.2 1.860.8 21.963.8 12.262.t 0.df p-value 10 0.92 n-back 1-back 2-back 3-back Pre- n3 PUFA 0.9860.04 0.9360.10 0.6560.27 Post- n3 PUFA 0.9960.02 0.9460.09 0.8060.29.89 10 ,0.01 26.30 10 ,0.01 20.13 10 0.90 3.49 10 0.tdfp-value 0.17 0.70 0.21.480 10 20.399 10 22.292n? PUFALA AAValues are mean and standard deviation (SD), n = 11 per condition. p-values are from two-tailed, paired t tests; t is t statistic; df is degrees of freedom. doi:10.1371/journal.pone.0046832.tValues are mean and standard deviation (SD), n = 11 per condition. p-values are from two-tailed, paired t tests; t is t statistic; df is degrees of freedom. doi:10.1371/journal.pone.0046832.tOmega-3 Fatty Acid Supplementation and VMATFigure 2. Shows the relationship between pre-supplementation RBC DHA or EPA in x-axis and pre-supplementation performance (AHR) in 3-back test in y-axis. The AHR ranges from 1 (best performance) to 21 (worst performance), with a score of 0 corresponding to performance at chance level. RBC DHA (Panel A), but not EPA (Panel B) was associated with performance in the task. doi:10.1371/journal.pone.0046832.gsubjects) and 20.0160.14 in the high.
