## 4 Discussion  
This study evaluated the differences in plasma free omega-3 PUFAs in relation to _FADS2_ gene variants in a group of overweight children, taking into account their respective dietary intakes. The results showed that the low-frequency G allele of rs526126 was associated with higher levels of plasma DHA only for DHA and EPA intakes lower than 10.56 mg and 3.45 mg, respectively. The basic steps of the n-3 LCPUFAs synthesis are shown in Figure 2.  
Figure 1: Distribution of plasma-free DHA levels grouped by _FADS2_ rs526126 for the entire group (**A**), grouped according to EPA (**B**) and DHA (**C**) dietary intakes. Mann–Whitney test was used to assess statistical significance between groups, the brackets marked with * indicating the two groups for which these differences were identified. Horizontal lines within each boxplot indicate minimum, first quartile (Q1), median, third quartile (Q3), and maximum. Legend: DHA, docosahexaenoicenoic acid; EPA, eicosapentaenoic acid; _FADS2_, fatty acid desaturase 2 gene. Since only six subjects had the GG genotype overall, out of which only three participants had complete datasets, the data for GG genotype were not included in the statistical analysis for dietary intakes-grouped datasets. Initially, data grouping according to dietary PUFAs intakes was made by quartiles, results indicating that the differences in plasma DHA levels between _FADS_ rs526126 genotypes were found within ALA intakes Q1 and Q2 and DHA Q3. Having established that plasma DHA was influenced mainly in lower quartiles, grouping for genotype-based analysis was then performed using EPA and DHA medians as cut-off values. In previous studies, rs526126 was found to be associated with lower D5 desaturase activity, while the other SNPs in the _FADS2_ gene were found to be associated with higher D6 desaturase activity [23]. However, no effect on D4 desaturase activity was observed. Until now, only in-vitro studies have demonstrated that _FADS2_ can regulate D4 desaturation to yield DHA [24; 25]. Interestingly, neither ARA levels nor the ratio between LA and ARA was associated with _FADS2_ variants, suggesting that no significant D6 desaturation occurred in this case (data not shown). In contrast to the previous reports [26], we did not find any association between rs526126 variant and C-reactive protein (CRP) levels, nor between any of the investigated free PUFAs and CRP (data not shown). Our data indicated that DHA plasma levels were more closely associated with rs526126 than EPA plasma levels. This can be explained by the fact that DHA is the final product of the desaturation cascade and thus is more likely to be influenced by _FADS2_ activity. Figure 2: ALA, alpha–linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; _FADS2_, fatty acid desaturase 2 gene; _FADS1_, fatty acid desaturase 1 gene; _ELOVL2_, fatty acid elongase 2; _ELOVL5_, fatty acid elongase 5. Moreover, the plasma levels of free DHA were higher than the ALA and EPA, leading to the conclusion that a significant amount of DHA is synthesized endogenously, in addition to their respective intakes. Differences were even more evident when the EPA and DHA dietary intakes were low, leading to the hypothesis that _FADS2_ activity is dependent on them and that the presence of rs526126 low-frequency allele (G) may result in increased desaturation activity. Esterified FAs from plasma and red blood cells were also investigated, but no association with _FADS2_ variants was found (data not shown), which may indicate that _FADS2_ action on n-3 can only be observed over short periods of time. In addition, the fact that the blood was collected after overnight fasting may indicate that the _FADS2_ acts on n-3 PUFA only when the substrate competition is low. Furthermore, this study aligns with the previously reported fact that FA plasma composition can be used as an indicator of recent fat intake [27].