Hepatic PUFA biosynthesis in pregnancy

Pregnancy involves increased demands on the mother for PUFA, to meet both her requirements and those of her fetus. The human fetus accumulates 22 : 6n-3, beginning in the second trimester, particularly into the central nervous system⁽ Reference Innis ^{13 ,} Reference Kuipers, Luxwolda and Offringa ^{36 )} and deficits in 22 : 6n-3 incorporation into developing brain have been associated with cognitive deficits in non-human primates⁽ Reference Reisbick, Neuringer and Hasnain ^{37 )}. The concentration of 22 : 6n-3, but not 20 : 4n-6, has been shown to increase in plasma phosphatidylcholine (PC) during the second trimester of pregnancy, specifically an increase in PC16:0/22:6⁽ Reference Postle, Al and Burdge ^{38 )}, which precedes the period of prenatal 22 : 6n-3 accumulation into fetal brain⁽ Reference Kuipers, Luxwolda and Offringa ^{36 )}. One interpretation is that such adaptation of maternal liver lipid metabolism may facilitate adequate supply of 22 : 6n-3 to the fetal brain⁽ Reference Postle, Burdge and Al ^{39 )}. Total plasma 22 : 6n-3 concentration has also been shown to increase between 18 and 29 d post-conception in women undergoing assisted reproduction⁽ Reference Meyer, Onyiaodike and Brown ^{40 )}. This change in plasma PC composition has been shown in rats to involve changes in the composition of the maternal hepatic diacylglycerol pool destined for PC synthesis and decreased acyl remodelling of sn-1 16 : 0 to sn-1 18 : 0 PC molecular species⁽ Reference Burdge, Hunt and Postle ^{41 )} and to be accompanied by increased liver Fads1 and Fads2 mRNA expression⁽ Reference Burdge, Slater-Jefferies and Grant ^{29 ,} Reference Childs, Hoile and Burdge ^{42 )}, possibly by the action of progesterone⁽ Reference Childs, Hoile and Burdge ^{42 )}.

Despite evidence of coordinated changes in maternal hepatic PUFA metabolism, the contribution, if any, of such adaptations to meeting maternal and fetal demands for PUFA is not known. In pregnant women, maternal plasma PC 22 : 6n-3 concentration tracks during gestation such that the level in early pregnancy (11–17 weeks) predicts 21 % of the variation in late pregnancy (35 weeks)⁽ Reference Crozier, Sibbons and Fisk ^{43 )}. Therefore, it may be expected that position in the rank order of maternal 22 : 6n-3 status, which is a proxy for 22 : 6n-3 availability to the developing fetus, would influence the cognitive function of the child. However, maternal plasma 22 : 6n-3 concentration during pregnancy has not been associated with intelligence quotient (IQ) or markers of executive cognitive function at ages 4 or 6–7 years⁽ Reference Crozier, Sibbons and Fisk ^{43 ,} Reference Brouwer-Brolsma, van de Rest and Godschalk ^{44 )}, cognition at 4 years⁽ Reference Ghys, Bakker and Hornstra ^{45 )} or 7 years⁽ Reference Bakker, Ghys and Kester ^{46 )}, nor with problem behaviour at 7 years⁽ Reference Krabbendam, Bakker and Hornstra ^{47 )}. Moreover, 22 : 6n-3 concentration has been shown to be approximately 32 % lower in umbilical cord blood and 62 % lower in breast milk from vegetarian pregnancies compared with omnivores⁽ Reference Sanders and Reddy ^{48 )} which suggests that metabolic adaptations that increase 22 : 6n-3 concentration in maternal blood during pregnancy are insufficient to compensate for low intake of 22 : 6n-3 in vegetarians⁽ Reference Burdge, Tan and Henry ^{49 )}. One possibility is that maternal adaptations to hepatic PUFA metabolism may be involved in the adjustment of the mother’s tissues to pregnancy rather than contributing directly to the PUFA supply and development of the fetus. For example, in the rat increased maternal plasma PC16:0/22:6 concentration occurs in late pregnancy and may facilitate supply of 22 : 6n-3 for incorporation into milk⁽ Reference Burdge, Hunt and Postle ^{41 )}.

PUFA biosynthesis in leucocytes

Leucocytes can convert 18 : 3n-3 and 18 : 2n-6 to longer-chain, more unsaturated PUFA, although the extent of such conversion appears to differ between types of immune cells and with the activation state of the cells. Radiolabelled 18 : 2n-6 can be converted to 20 : 2n-6 in quiescent murine macrophages without detectable further desaturation or elongation⁽ Reference Chapkin, Somers and Erickson ^{69 )}. Incubation of murine peritoneal macrophages with the calcium ionophore A23187, phorbol myristate acetate, or zymosan did not induce further interconversion of 20 : 2n-6⁽ Reference Chapkin, Somers and Erickson ^{69 )}. Furthermore, [¹⁴C]18 : 3n-6 can be converted to 20 : 3n-6, but not to 20 : 4n-6, in stimulated murine macrophages⁽ Reference Chapkin and Coble ^{66 )}. Absence of detectable 20 : 4n-6 suggests that murine macrophages lack Δ5 desaturase activity.

In contrast to macrophages, mitogen stimulation increased the proportions of 18 : 1n-9, 22 : 5n-3 and 22 : 6n-3 and decreased the proportion of n-6 PUFA in T-cell membranes⁽ Reference Anel, Naval and Gonzalez ^{70 )}. This suggests remodelling of membrane phospholipids and/ or increased synthesis or uptake of specific fatty acids including longer-chain n-3 PUFA. Mitogen stimulation of human PBMC increased Δ9, Δ6 and Δ5 desaturase activities, which was associated with increased interconversion of [¹⁴C]18 : 0 to 18 : 1n-9 and of [¹⁴C]18 : 2n-6 and [¹⁴C]18 : 3n-3 to triene and tetriene PUFA⁽ Reference Anel, Naval and Gonzalez ^{71 )}. The authors noted that it was uncertain whether such capacity was sufficient to account for the changes in membrane composition that accompany T-cell activation. Moreover, incubation of human PBMC with 18 : 2n-6 or 18 : 3n-3 followed by mitogen stimulation increased the incorporation of these fatty acids into the cells⁽ Reference Anel, Naval and Gonzalez ^{71 )}. Incubation of mitogen-stimulated rat lymph node lymphocytes with 18 : 3n-3 increased the proportions of 18 : 3n-3, 20 : 5n-3 and 22 : 5n-3 but not 22 : 6n-3 in membrane phospholipids⁽ Reference Calder, Yaqoob and Harvey ^{72 )} which suggests that at least some of the changes in membrane composition in activated T cells may involve endogenous PUFA synthesis, while other, such as increased 22 : 6n-3 content, may represent selective uptake from their environment. Although incubation with 18 : 2n-6 increased the proportion of this fatty acid in T-cell phospholipids, there was only a small, non-significant increase in the 20 : 4n-6 content⁽ Reference Calder, Yaqoob and Harvey ^{72 )}. The apparent inconsistency in the conversion of 18 : 3n-3 and 18 : 2n-6 was not explained by the authors. These findings suggest that, in contrast to macrophages, human and rat lymphocytes express Δ6 and Δ5 desaturase, and that elongase expression is up-regulated in activated cells. Increased membrane fluidity induced by increasing the proportion of unsaturated fatty acids in membrane phospholipids has been suggested a possible mechanism by which unsaturated fatty acids could inhibit immune cell function by modifying the membrane protein activity⁽ Reference Calder, Yaqoob and Harvey ^{72 )}. Moreover, increasing the synthesis of PUFA substrates may facilitate the production of pro-resolving mediators and hence attenuate the immune response⁽ Reference Halade, Black and Verma ^{73 )}.

A recent study of PUFA biosynthesis in human PBMC confirmed that mitogen stimulation induced increased incorporation of [¹³C]18 : 3n-3 into cell lipids and conversion to longer-chain, more unsaturated fatty acids⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}. This was accompanied by marked up-regulation of FADS2, FADS1, ELOVL5 and ELOVL4 mRNA expression⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}. The first two reactions were chain elongation of 18 : 3n-3 to 20 : 3n-3 by an unidentified elongase followed by Δ8 desaturation of 20 : 3n-3 to 20 : 4n-3, essentially the reverse of the initial reactions of the pathway reported in liver⁽ Reference Voss and Sprecher ^{14 ,} Reference Sibbons, Brenna and Lawrence ^{34 )} (Fig. 2). The first Δ6 desaturation in liver has been shown to be rate limiting⁽ Reference Rodriguez, Sarda and Nessmann ^{74 )} and to preferentially catalyse conversion of 18 : 3n-3 compared with 18 : 2n-6⁽ Reference Rodriguez, Sarda and Nessmann ^{74 )}. Thus, one implication of the apparent substitution of the initial Δ6 desaturation with elongase activity is that the regulation of the PUFA synthesis pathway in PBMC may differ from that of other tissues. For example, elongases have been shown to differ in substrate preference in terms of chain length and level of unsaturation such that elongase 5 catalyses elongation of 18- and 20-carbon PUFA, while elongase 2 exhibits marked preference of 20- and 22-carbon PUFA⁽ Reference Gregory, Gibson and Cook-Johnson ^{12 )}. Both of these enzymes preferentially catalyse conversion of n-3 compared with equivalent n-6 PUFA⁽ Reference Gregory, Gibson and Cook-Johnson ^{12 )}. If so, this may have implications for understanding the influence of dietary fatty acids on immune function. The protein product of the baboon Fads2 gene has been associated with Δ8 desaturase activity when transfected into yeast cells, although Δ6 desaturase activity predominated⁽ Reference Park, Kothapalli and Lawrence ^{75 )}. Thus, it is possible that Δ8 desaturase activity in PBMC was also associated with the protein encoded by the FADS2 gene, although no Δ6 desaturase activity was detected⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}. Moreover, Δ4-desaturase activity has been demonstrated in MCF7 breast cancer cells transfected with FADS2 that do not normally express this gene⁽ Reference Park, Park and Kothapalli ^{17 )}. Thus, the desaturase activity of the FADS2 protein product may depend upon the cellular environment in which it is expressed. ELOVL2 was not expressed in PBMC and the PUFA synthesis pathway was truncated after synthesis of 22 : 5n-3⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}.

Fig. 2 The proposed pathway for PUFA biosynthesis linked to proliferation of T lymphocytes⁽ Reference Cho, Nakamura and Clarke ^{57 )}. Dashed arrows indicate putative reactions. VLCPUFA, very long-chain PUFA.

ELOVL4 catalyses the conversion of ≥C20 PUFA, but not 22 : 6n-3, to PUFA with chain length C24–C38⁽ Reference Agbaga, Brush and Mandal ^{76 –} Reference Suh and Clandinin ^{79 )}, which are involved in the formation and structure of lipid rafts required for T-cell signalling⁽ Reference Iwabuchi, Nakayama and Iwahara ^{80 )}. Thus, it has been suggested that truncation of the PUFA synthesis pathway after 22 : 5n-3 and up-regulation of ELOVL4 may provide very long-chain PUFA required for T-cell activation⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}. Moreover, a lipoxygenase-derived metabolite of 20 : 3n-9 can modulate leukotriene B₄ synthesis, and hence neutrophil activation, via inhibition of leukotriene A hydrolase⁽ Reference Cleland, James and Proudman ^{81 )}. It is not known whether 20 : 3n-3 can also be converted into bioactive metabolites but demonstration of such activity may suggest an additional mechanism to link Δ8 desaturation to immune function. Of potential importance to understanding the role of PUFA synthesis in stimulated T cells, pharmacological inhibition of the FADS2 protein reduced mitogen-induced T-cell proliferation⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}. Thus, PUFA synthesis appears to be involved in the regulation of T-cell proliferation possibly by providing substrates for the assembly of lipid microdomains and/or through the formation of novel lipid mediators (Fig. 2).

PUFA synthesis in cancer cells

PUFA synthesis has been described in several cancer cell types. One recent study compared PUFA synthesis in Jurkat T-cell leukaemia cells with PBMC⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}. The findings showed that interconversion of [¹³C]18 : 3n-3 was constitutive and 17-fold greater in Jurkat cells than in mitogen-stimulated PBMC. FADS2, FADS1, ELOVL4 and ELOVL5 expression was also up to 17-fold greater in Jurkat cells than in stimulated PBMC, and ELOVL2 was expressed which is consistent with the conversion of 18 : 3n-3 to 22 : 6 in Jurkat cells. These findings suggest that oncogenic transformation may disrupt PUFA synthesis at the level of transcription at several points in the pathway. The major product of PUFA synthesis in Jurkat cells was 22 : 5n-3 rather than 20 : 3n-3, which was the major fatty acid synthesised from 18 : 3n-3 in PBMC. Jurkat cells expressed both Δ8 and Δ6 desaturase activities, while PBMC only expressed Δ8 desaturase activity. Furthermore, inhibition of the FADS2 protein did not significantly alter Jurkat cell proliferation⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}. Stimulation of PBMC induced hypermethylation of a 600-bp region of the FADS2 promoter. In contrast, this putative regulatory region was hypomethylated in Jurkat cells⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}. Together, these findings suggest that PUFA synthesis and its role in regulating lymphocyte cell proliferation is disrupted in Jurkat cells.

The activity of Δ6 desaturase and the expression of FADS2 mRNA were shown to be substantially greater in murine xenografts of B16 melanoma and Lewis lung carcinoma (LLC) cells compared with the normal tissue⁽ Reference He, Qu and Wan ^{82 )}. FADS2 RNAi knockdown inhibited proliferation and reduced tumour size in both B16 and LLC tumours⁽ Reference He, Qu and Wan ^{82 )} which suggests that PUFA synthesis may be involved in the regulation of mitosis in these cells. The ratio of the sum of n-6 PUFA to 18 : 2n-6 in primary breast cancer tissue, a proxy marker of PUFA synthesis, was greater than that in uninvolved tissues⁽ Reference Pender-Cudlip, Krag and Martini ^{83 )} and higher in more aggressive oestrogen receptor-negative (ER–) tumours compared with ER+ tumours⁽ Reference Pender-Cudlip, Krag and Martini ^{83 )}.

The absence of FADS2 expression in MCF7 breast carcinoma cells results in the synthesis by Δ5 desaturase of 5,11,14–20:3 and 5,11,14,17–20:4 which lack the 8–9 double bond of their eicosanoid substrate counterparts 20 : 4n-6 and 20 : 5n-3⁽ Reference Park, Kothapalli and Lawrence ^{84 )}. This suggests a mechanism by which deletion of FADS2 during malignant transformation may lead to disruption of cellular signalling and the production of novel second messengers⁽ Reference Park, Kothapalli and Lawrence ^{84 )}. Furthermore, activation of the c-Ha-ras oncogene, but not the c-Ha-ras pro-oncogene, in spontaneously immortalised MCF10A mammary epithelial cells resulted in the loss of capacity for Δ6 and Δ4 desaturation⁽ Reference Grammatikos, Harvey and Subbaiah ^{85 )}.

Together these studies suggest that altered regulation of PUFA synthesis is a characteristic of at least some types of cancer, which may be linked to their proliferative capacity. This is consistent with the findings of studies that show reduction in proliferation and/or induction of apoptosis of cancer cells treated with 22 : 6n-3⁽ Reference Liu and Ma ^{86 –} Reference Burdge, Rodway and Kohler ^{89 )}.

PUFA synthesis in spermatogenesis

Human sperm is highly enriched in 22 : 6n-3. Morphologically normal sperm cells contain approximately 35 % 22 : 6n-3, while the proportion of 20 : 4n-6 is approximately 10·5 %⁽ Reference Lenzi, Picardo and Gandini ^{90 )}. The presence of 22 : 6n-3 in sperm membrane phospholipids has been associated with cell motility, membrane fusion and the synthesis of second messengers and is consistent with the presence of high levels of antioxidant defence mechanisms⁽ Reference Lenzi, Picardo and Gandini ^{90 )}. Experimental EFA deficiency in rats can induce degeneration of seminiferous tubules and reduced sperm formation and fertility⁽ Reference Leath, Northop and Harrison ^{91 )}. Moreover, feeding male Fads2 null mice a diet containing 18 : 3n-3 and 18 : 2n-6, without longer-chain PUFA, induced 74 % reduction in the proportion of 22 : 6n-3 and 92 % decrease in the relative amount of 20 : 4n-6 (92%) in testis⁽ Reference Roqueta-Rivera, Stroud and Haschek ^{92 )}. This was accompanied by arrested spermatogenesis and degradation of seminiferous tissue⁽ Reference Roqueta-Rivera, Stroud and Haschek ^{92 )}. Dietary supplementation of Fads2 null mice with 22 : 6n-3 restored spermatogenesis and fertility to levels equivalent to wild-type mice⁽ Reference Roqueta-Rivera, Stroud and Haschek ^{92 )}. This suggests that PUFA synthesis is important for fertility in mice fed a diet without longer-chain PUFA. However, because the knockout was not tissue-specific, it is not possible to conclude whether the activity of the PUFA synthesis pathway in testis is involved. However, there is some evidence for local PUFA synthesis in testis. Microsomes from human testis can convert [¹⁴C]18 : 2n-6 to 20 : 3n-6 (88 %) and 20 : 4n-6 (12 %) but not 18 : 3n-6⁽ Reference Albert, Rhamy and Coniglio ^{62 )}, indicating that the initial reactions were elongation of 18 : 2n-6 to 20 : 2n-6, followed by Δ8 desaturation to form 20 : 3n-6 followed by Δ5 desaturation to 20 : 4n-6. This pathway has also been reported in rat testis⁽ Reference Albert and Coniglio ^{93 )}. Moreover, the proportion of 22 : 6n-3 in testis from humans and from Rhesus macaques increases during puberty and remains essentially unchanged during adulthood⁽ Reference Coniglio, Grogan and Rhamy ^{94 ,} Reference Connor, Lin and Neuringer ^{95 )}. One possible mechanism is that testicular 22 : 6n-3 synthesis increases during puberty to meet the requirements for 22 : 6n-3 during spermatogenesis. If so, this suggests that regulation of PUFA synthesis in testis differs from hepatocytes in which testosterone has no significant effect on 22 : 6n-3 synthesis⁽ Reference Childs, Kew and Finnegan ^{35 )}. However, the contribution of testicular PUFA synthesis to male fertility has yet to be determined.

PUFA synthesis in arteries

Bovine aortic endothelial cells have been shown to retroconvert 22 : 6n-3 to 22 : 5n-3 and 20 : 5n-3⁽ Reference Hadjiagapiou and Spector ^{96 )} and 22 : 4n-6 to 20 : 4n-6⁽ Reference Mann, Kaduce and Figard ^{97 )}. Elongation of [¹⁴C]20 : 5n-3 to 22 : 5n-3 and of [¹⁴C]20 : 4n-6 to 22 : 4n-6 has also been shown in human vascular endothelial cells⁽ Reference Rosenthal, Garcia and Jones ^{63 )}, although the role of PUFA synthesis in endothelial cell function has not been reported. Phenylephrine stimulation of isolated rat aortae increased Fads1 and Fads2 mRNA expression compared with unstimulated vessels⁽ Reference Kelsall, Hoile and Irvine ^{60 )}. Moreover, treatment of isolated rat aortae or human femoral artery with the FADS2 protein inhibitor SC26196 or rat aortae with the Δ5 desaturase inhibitor sesamin reduced phenylephrine-induced vasoconstriction and secretion by rat aortae of pro-constriction eicosanoids PGF_2α , PGE₂ and thromboxane A2⁽ Reference Kelsall, Hoile and Irvine ^{60 )}. Thus, PUFA synthesis, which was localised to VSM rather than the endothelium⁽ Reference Kelsall, Hoile and Irvine ^{60 )}, appears to be involved in regulating vasoconstriction. Mouse aortae, immortalised murine VSM (MOVAS) cells and human primary aortic VSM cells express Fads1, Fads2 and Elovl5 but not Elovl2 ⁽ Reference Irvine, Lillycrop and Fielding ^{61 )}. This is consistent with the apparent absence of conversion of 22 : 4n-6 to 22 : 5n-6, in MOVAS cells⁽ Reference Irvine, Lillycrop and Fielding ^{61 )}. Since Elovl2 was also not expressed in PBMC, it is possible that suppression of Elovl2 transcription is a characteristic of at least some excitable tissues although the precise function may differ. The absence of Elovl2 expression did not appear to involve hypermethylation of the promoter⁽ Reference Irvine, Lillycrop and Fielding ^{61 )}. Inhibition of Δ6 or Δ5 desaturase significantly reduced phenylephrine-mediated Ca release and secretion of PGE₂ and PGF_2α in MOVAS cells⁽ Reference Cho, Nakamura and Clarke ^{57 )}. The findings suggest that α ₁-adrenergic receptor-mediated vasoconstriction involves activation of PUFA synthesis acting via regulation of endogenous Ca²⁺ release and secretion of specific vaso-active eicosanoids. One possible mechanism that has been suggested to explain the link between PUFA synthesis and vasoconstriction is activation of protein kinase C _ζ and inhibition of myosin light chain phosphorylase by newly synthesised 20 : 4n-6⁽ Reference Irvine, Lillycrop and Fielding ^{61 )}.

Polymorphisms in genes involved in PUFA biosynthesis

The region of human chromosome 11 that contains the FADS1 and FADS2 genes in a head-to-head orientation (11q12.2–q13.1), together with FADS3⁽ Reference Schaeffer, Gohlke and Muller ^{98 )}, contains 4391 variants, of which 217 can result in amino acid changes⁽ Reference Minihane ^{99 )} and hence potentially alter enzyme activity. This region has been identified as a cancer ‘hotspot locus’⁽ Reference Park, Kothapalli and Lawrence ^{84 ,} Reference Marquardt, Stohr and White ^{100 )}. Several genome–wide association studies have reported associations between polymorphisms in genes associated with PUFA synthesis and the concentrations of PUFA in blood. A meta-analysis of 8866 individuals from five cohorts showed that minor alleles of SNP in FADS2 and FADS1 were associated with a higher level of 18 : 3n-3 and a lower level of 20 : 5n-3, 22 : 5n-3 and 22 : 6n-3, while minor alleles of ELOVL2 were associated with higher proportions of 20 : 5n-3 and 22 : 5n-3 and a lower proportion of 22 : 6n-3⁽ Reference Lemaitre, Tanaka and Tang ^{101 )}. The variation in the proportions of n-6 PUFA in plasma phospholipids explained by eleven locus haplotypes has been shown to be 18 : 2n-6, 9·2 %; 18 : 3n-6, 7·9 %; 20 : 2n-6, 12·3 %; 20 : 3n-6, 10·8 % and 20 : 4n-6, 28·5 %⁽ Reference Mann, Kaduce and Figard ^{97 )}. In contrast, there were only limited associations with n-3 PUFA (18 : 3n-3, 5·4 %; 20 : 5n-3, 6·9 %; 22 : 5n-3, 5·1 %; 22 : 6n-3, 2·9 %)⁽ Reference Schaeffer, Gohlke and Muller ^{98 )}. However, whether these polymorphisms alter tissue function is not known. Moreover, interpretation of such associations may be confounded by contributions of hepatic phospholipid biosynthesis by the cytosine diphosphate choline and phosphatidylethanolamine (PE) N-methylation pathways to the fatty acid composition of plasma phospholipids because human⁽ Reference Pynn, Henderson and Clark ^{102 )} and rat⁽ Reference Burdge, Hunt and Postle ^{41 )} liver selectively synthesise 20 : 4n-6- and 22 : 6n-3-containing PC by PE N-methylation. One possible explanation for the greater effect of FADS polymorphisms on the proportions of n-6 PUFA for n-3 PUFA is the typically greater intake of 18 : 2n-6 than 18 : 3n-3⁽ Reference Burdge and Calder ^{103 )} and consequently greater flux of n-6 PUFA through the PUFA synthesis pathway than 18 : 3n-3. Women carrying FADS1 and FADS2 minor alleles had lower proportions of 20 : 4n-6 and 22 : 5n-3 in their plasma and in breast milk, although the effect on the development of their infants and children was not assessed⁽ Reference Xie and Innis ^{104 )}. Thus, mutations in genes involved in PUFA synthesis are associated with variations in plasma PUFA status, which suggests that hepatic PUFA synthesis can contribute to the levels of circulating PUFA. However, these findings have not been used to estimate the magnitude of this contribution. Carriers of the minor allele (deletion) of a 22 bp FADS2 insertion–deletion mutation had lower expression of FADS1 in lymphocytes⁽ Reference Reardon, Zhang and Kothapalli ^{105 )} and lower 20 : 4n-6 status compared with the major insertion allele⁽ Reference Kothapalli, Ye and Gadgil ^{106 )}. The frequency of the insertion/insertion allele was greater in populations with a tradition of vegetarian diet compared with Western omnivorous populations who had a higher frequency of the deletion/deletion genotype. This has been suggested as a possible adaption to low intakes of pre-formed longer-chain PUFA⁽ Reference Kothapalli, Ye and Gadgil ^{106 )}.

To date, the effect of polymorphisms in genes involved in PUFA synthesis on tissue function has not been investigated directly, although such studies may provide novel insights into tissue function and disease aetiology. However, there is some evidence that individuals carrying minor FADS1–FADS2 alleles that were associated with lower 20 : 4n-6 status have a lower prevalence of allergic disease which may be attributable to a reduction in the availability of 20 : 4n-6 for the synthesis of pro-inflammatory eicosanoids⁽ Reference Schaeffer, Gohlke and Muller ^{98 )}. However, since PUFA synthesis can regulate T-cell activation and proliferation⁽ Reference Sibbons, Irvine and Pérez-Mojica ^{59 )}, it is also possible that reduced desaturase activity may constrain the activity of the PUFA synthesis pathway, thus limiting cell proliferation and the immune response.