EFA FUNCTIONS

III. EFA FUNCTIONS

A. Membrane Protein Boundary Lipids

As noted in the previous section, most fatty acids in the body are present as phospholipids in biological membranes, and the fatty acyl moieties of these phospholipids determine many of the functional and biochemical characteristics of those membranes. Embedded in the lipid matrix of a biological membrane are the proteins that confer specificity to that particular membrane. In the outer segments of the rod cells of the retina, for example, there are a series of pancakelike stacks of membranes in which the retinal-binding protein, rhodopsin, is found (Bazan and Rodriguez de Turco, 1994; Gordon and Bazan, 1990) ( Fig. 5 ). The concentration of rhodopsin in these membranes was recently shown to be dependent on the DHA content of the phospholipids comprising those membranes (Suh et al., 1997). That is, there appears to be a positive correlation between rhodopsin density (a characteristic that should define the light sensitivity of the eye) and DHA content of the retina. Furthermore, if an animal is made omega-3 deficient and the DHA levels are drastically reduced, the DHA is replaced by omega-6 DPA. Under such conditions, the visual acuity of the animal is compromised (Neuringer et al., 1986). Thus, the existence of an additional double bond at the ∆19 position of an otherwise identical molecule has

a dramatic effect on the performance of a specific organ (Salem and Niebylski, 1995;

Figure 4 Complex lipid forms.

a pivotal role in the ‘‘boundary lipid’’ of a particular membrane protein such that any alteration in the boundary lipid results in a significant impact of the functioning of that protein. Across 500 million years of evolution DHA—not DPA—has been selected as providing the optimal lipid environment for this photoreceptor pigment protein (Broadh- urst et al., 1998).

There are many other examples of how specific fatty acids are required for the optimal function of membrane proteins. This is not unexpected because the orientation of a phospholipid in the membrane is significantly impacted by the degree of unsaturation of its fatty acid moieties. A single cis-double bond confers a 37 ° kink in the orientation of a fatty acid. A second double bond imparts a second bend in the structure and so on.

A molecule with six double bonds, such as DHA, can have a helical configuration as a

Figure 5 Retinal rod outer segments.

membrane ( Fig. 1 ). Such a molecule has a much larger cross sectional volume than, for example, a simple saturated fatty acid like palmitic or stearic acid and has many very specific packing arrangements that can optimize the function of certain membrane proteins.

B. Eicosanoid Functions

In addition to their role as structural entities affecting the performance of membrane- bound proteins, the twenty carbon metabolites of the essential fatty acids have important roles in the body as circulating eicosanoids (Holman, 1986; Lagarde, 1995; Lands, 1989). The complexity of the biochemical conversions are shown in Fig. 6 for arachidonic acid only. Similar metabolic fates occur in the other twenty-carbon PUFAs such as EPA and dihomo-gamma linolenic acid [DGLA; C20:3( ∆9.12.15)]. These eicosanoids have a rela- tively high specificity in the body to elicit a biological response, and are therefore un- der remarkably tight regulation. Prostaglandin E2 (PGE2), for example, is a potent elicitor of platelet aggregation and vasoconstriction (Holman, 1986). The body’s response to wounding involves the release of free ARA, the elevation of PGE2, and the subsequent constriction of blood flow around the wound. This is an important, acute survival mecha- nism. In the longer term, however, it could lead to elevated blood pressure and an increased risk of coronary vascular disease. However, another ARA metabolite, prostacyclin I2, has

a strong effect on inhibiting platelet aggregation, so that there may be a considerable latitude of PGE2 levels which provides the organisms with a rapid response mechanism with little, or no, long-term effects.

Many of the eicosanoids have a direct influence on biological responses associated with immune function. These include the inflammatory response as well as induction of macrophages and production of antibodies in response to some challenge to the organism. In general, the omega-6 eicosanoids have been considered as proinflammatory and up- regulators of typical immunological responses. In a recent well-controlled human feeding trial using an ARA-rich triglyceride providing about 1.5 g of ARA per day (over 50 days), both thromboxane B2, and a metabolite of PGI2, were shown to increase, by 41% and 27%, respectively (Ferretti et al., 1997). Although this elicited no measurable change in platelet aggregation or bleeding time (Nelson et al., 1997a), the researchers reported an

Figure 6 Eicosanoids produced from arachidonic acid and their function.

elevation in antibody titre in response to a heat-killed influenza virus challenge, suggesting an up-regulation of the immune response (Kelley et al., 1997). On the other hand, fish oil feeding trials which provide high levels of the omega-3 eicosanoid precursor EPA, have reported a significant decrease in platelet aggregation and increase in bleeding time (Cobiac et al., 1991; Eritsland et al., 1989; Mark and Sanders, 1994; Ward and Clarkson, 1985), a down-regulation of the immune response (Meydani et al., 1993) and a general anti-inflammatory response (Lee et al., 1991). Such a response is particularly useful in patients with proinflammatory disorders such as rheumatoid arthritis or asthma (Kremer et al., 1990; Lee et al., 1991; Raederstorff et al., 1996).

C. Signalling Pathways

Although EFAs have generally been considered important because of their nutritional value, in recent years it has become clear that these fatty acids may also play an important role in gene regulation (Clarke and Jump, 1994; Sesler and Ntambi 1998). Dietary fats, particularly PUFAs, exhibit a general effect on expression of genes in lipogenic tissues. High levels of PUFAs result in a decrease of activity of liver enzymes involved in lipogen- esis, and PUFA restriction appears to induce the expression of lipogenic enzymes. This response does not seem to be specific to either omega-6 or omega-3 LC-PUFAs (Sesler and Ntambi, 1998). Recent studies have identified similar responses in adipose tissue although these may be more class specific. Fatty acid synthetase and lipoprotein lipase expression in adipose tissue of the rat has been shown to be specifically activated by omega-3 LC-PUFAs (Cousin et al., 1993).

Polyunsaturated fatty acid regulation of gene expression in nonlipogenic tissues has also been reported. These genes include Thy-1 antigen on T-lymphocytes, L-fatty acid binding protein, apolipoproteins A-IV and C-III, sodium channel gene in cardiac myo- cytes, acetyl Co-A carboxylase in pancreatic cells, and steaoryl-CoA desaturase in brain tissue (Gill and Valivety, 1997; Sesler and Ntambi, 1998).

The reported stimulation of superoxide dismutase by LC-PUFAs (Phylactos et al., 1994) represents a mechanism whereby intracellular antioxidants may be stimulated. This may be particularly relevant in neonatology as infants which normally receive a good endogenous supply of DHA from their mother (across the placenta prenatally or via breast milk postnatally) may have a better antioxidant status than those infants fed a formula without supplemental DHA and ARA. Breast-fed infants certainly appear to be protected from certain oxidant-precipitated pathologies such as narcotizing enterocolitis (NEC) (Crawford et al., 1998) and this has been correlated to the higher levels of cupric/zinc superoxide dismutase in the erythrocytes of breast-fed versus formula-fed infants (Phy- lactos et al., 1995). Such an improvement of intracellular antioxidants may also explain

a recent observation that feeding preterm infants with a DHA/ARA-supplemented formula results in an 18% decrease in the incidence of NEC (Carlson et al., 1998). The molecular mechanisms whereby, essential fatty acids can affect gene expression are still poorly understood. Some studies have suggested that the action of the PUFAs may be both at the level of transcription and others at the stabilization of the mRNA. A cis-acting PUFA responsive element (PUFA-RE) has been proposed to be in the promoter region of all PUFA-regulated genes, and a nuclear factor binding to this element has been demonstrated (Waters et al., 1997). Through an understanding of how dietary EFAs may affect gene regulation, especially with respect to lipogenesis and the production of intracel- lular antioxidants, we can make better use of supplemental EFAs in our diet to improve long-term health and well being.

D. DHA Function

DHA is somewhat of an enigma in the body. It is an energetically expensive molecule to synthesize, it requires a complicated biochemical route which includes many different gene products, it is prone to oxidative attack because of its abundance of double bonds, and yet is still a major component of many important organ systems in the body. Docosa- hexaenoic acid is found in close association with membrane proteins of the 7-transmem- brane structure (7-Tm), G-protein coupled receptors (i.e., serotonin receptors, acetylcho- DHA is somewhat of an enigma in the body. It is an energetically expensive molecule to synthesize, it requires a complicated biochemical route which includes many different gene products, it is prone to oxidative attack because of its abundance of double bonds, and yet is still a major component of many important organ systems in the body. Docosa- hexaenoic acid is found in close association with membrane proteins of the 7-transmem- brane structure (7-Tm), G-protein coupled receptors (i.e., serotonin receptors, acetylcho-

Leaf and colleagues have recently proposed that LC-PUFAs may play a role in calcium channel regulation (Billman et al., 1997; Xiao et al., 1997). In isolated cardiac myocytes, EPA and DHA are effective in blocking calcium channels, resulting in a restora- tion of normal rhythmicity to the isolated cells treated with ouabain (Leaf, 1995). In a dog model of sudden cardiac death (induced ventricular arrhythmia), Billman and colleagues (Billman et al., 1994, 1997) have shown that predosing animals with omega-3 LC-PUFAs significantly reduced the number of fatal arrhythmias or ventricular fibrillations. Similar results have been demonstrated with rat (McLennan et al., 1996) and primate models (Charnock et al., 1992). It is now believed that DHA may act as the endogenous calcium channel controlling factor in cardiac cells. The proposal drawn in Fig. 7 suggests such a mechanism in neural tissues, where an elevated intracellular calcium level stimulates a calcium-dependent phospholipase, which in turn cleaves off free DHA from the DHA- rich membranes. The local high concentration of free DHA then closes off the calcium channel, reducing the calcium inflow, and the internal calcium levels drop. Since it is DHA, not EPA that is enriched in neurological and cardiac cells, it is likely that DHA is

Figure 7 Role of DHA as a modulator of calcium channels.

the active fatty acid in this control mechanism. Indeed, McLennan and colleagues (1996) demonstrated that at low dietary intakes, it is DHA, not EPA, that inhibits ischaemia- induced cardiac arrhythmias in the rat. DHA also represents a safer control mechanism compared to twenty-carbon LC-PUFAs since the release of free eicosanoid precursors may result in a series of unwanted eicosanoid-related responses.