Los aspectos evaluados

Salmon contains micronutrients which could compensate for the nutritional deficiencies that are commonly observed in IBD subjects from poor dietary intake [162] or impaired nutrient absorption [163]. Of particular clinical relevance are deficiencies in calcium, vitamins D, B6, B12, and E [164, 165], all of which are present in salmon (Table 1.4). Several of these micronutrients have shown to suppress inflammation in rodents with experimental colitis. For example, supplementing diets with vitamin D and calcium

showed protective effects in Il10-/- _{mice (associated with tumor necrosis factor (TNF)}

pathway) [166] and selenium protected rats with experimental colitis [167].

Vitamin E is naturally present in salmon (mostly as α-tocopherol [168]) and as an antioxidant protects against lipid peroxidation in the muscle of the fish [169]. This antioxidant property of vitamin E could be useful in intestinal inflammation. n-3 PUFA are prone to peroxidation, and oxidised PUFA can activate transcription factors such as NFKB and subsequently trigger pro-inflammatory gene expression. A study has shown that diets supplemented with α-tocopherol protected rats from the oxidative stress associated with colitis [170]. Nevertheless, conflicting results regarding the anti- inflammatory and cancer-preventative features of tocopherols have been reported. The Selenium and Vitamin E Cancer Prevention Trial (SELECT) demonstrated that supplementation with α-tocopherol increased the risk of prostate cancer in healthy men [171, 172]. The authors further observed a 50% decrease of γ-tocopherol in plasma, concluding that the α-tocopherol supplementation may have reduced the beneficial effects of γ-tocopherol (potentially linked to bioavailability). While the tocopherol levels in

Table 1.4 Nutritional profile of farmed New Zealand Chinook salmon fillets (Oncorhynchus tshawytscha). Component Amount Energy 1334 kJ Protein 18.0 g Carbohydrate <1.0 g Fat 23.1 g Saturated 4.9 g Trans 0.05 g Monounsaturated 7.7 g Polyunsaturated 5.2 g Omega-3 3.1 g

Eicosapentaenoic acid (EPA) 1.0 g

Docosahexaenoic acid (DHA) 1.3 g

Docosapentaenoic acid (DPA) 0.5 g

Alpha linolenic acid (ALA) 0.2 g

Omega-6 2.1 g

Linoleic acid (LA) 1.7 g

Arachidonic acid (AA) <0.1 g

Elements Sodium 28 mg Potassium 374 mg Phosphorus 235 mg Magnesium 26 mg Calcium 9.2 mg Iron 0.2 mg Selenium 0.02 mg Iodine 0.005 mg Vitamins Vitamin A 0.06 mg Vitamin B1 (Thiamine) 0.15 mg Vitamin B2 (Riboflavin) 0.10 mg Vitamin B3 (Niacin) 6.79 mg Vitamin B6 0.47 mg Vitamin C 3.00 mg Vitamin D 0.02 mg Vitamin E 5.31 mg Vitamin B12 <0.01 mg

Table adapted from The King Salmon Company webpage [178], accessed 29th _{September 2011. Values}

1.4.2

Peptides

After digestion, peptides are either absorbed through the intestinal wall and/or affect the intestine locally [as reviewed in 179]. The antioxidant activity of fish protein hydrolysates (breakdown products of enzymatic conversion of fish proteins into smaller peptides [180]) has previously been shown [181-183] and potentially induce synergistic effects in combination with other nutrients [184, 185]. For example, Grimstad et al. [185] demonstrated that the expression of selected genes and pro-inflammatory cytokines (e.g. TNF or IL1B) were largely unaffected by dietary supplementation with hydrolysed salmon peptides or a fish oil diet in rats with DSS-induced colitis, but the combined supplementation of these fish products revealed synergistic effects on eicosanoid synthesis. While the fish oil diet increased the levels of EPA-derived eicosanoid prostaglandin E3 in the colon, the combined supplementation of fish oil and peptides further increased its levels. The authors linked this increase to elevated enzymatic activity of prostaglandin-endoperoxide synthase 2 (PTGS2, also called COX2), the enzyme that metabolises EPA to prostaglandin E3 [185].

1.4.3

Lipids

New Zealand Chinook salmon (Oncorhynchus tshawytscha) has a higher natural oil content than other salmon species [186, 187]. Fatty acids, comprising a hydrocarbon chain with a carboxyl moiety at one end and a methyl group at the other, are the major building block for complex lipids and can be saturated (no double bond between the carbon atoms), monounsaturated (one double bond (MUFA)), or polyunsaturated (two or more double bonds (PUFA)). The classification into n-3 and n-6 PUFA is based on the position of the first double bond starting from the methyl end of the hydrocarbon chain. Short-chain fatty acids (SCFA) refer to 19 or fewer carbon atoms, and long-chain to 20-

via several elongation and desaturation steps that mainly take place in the liver (Figure 1.6). The conversion of ALA to EPA is, however, limited and further conversion from EPA to DHA is even lower [188, 189]. The limiting steps in these conversions are the competition for the same enzymes in the n-6 pathway (metabolising the precursor n-6 PUFA linoleic acid (LA) to arachidonic acid (AA) (Figure 1.6)). Additionally, the activities of the enzymes depend on diet [80], hormones [190], and feedback inhibition by end products [191, 192]. Hence, direct DHA and EPA supplementation is more effective than de novo synthesis in increasing circulating LC n-3 PUFA concentrations.

Several studies have tested the anti-inflammatory potential of pure n-3 PUFA or fish oil (as a source of PUFA), however the results of these studies are inconsistent. Some

studies reported detrimental effects of fish oil diets on colitis in Il10-/- _{mice [193] or in}

infectious colitis mouse models [194]. Others have reported reduced colitis in response

to fish oil diets in TNBS-dosed rats [195] and in Il10-/- _{mice [196], in DSS-dosed rats fed}

a soybean/fish oil mixture [5] and in DSS-dosed rats fed an olive oil/fish oil mixture [7]. These studies were conducted with a mixture of DHA and EPA and observed effects cannot be specifically attributed to DHA or EPA. Some studies have reported potential

anti-inflammatory effects of pure EPA in Il10-/- _{mice [6] and DSS-dosed mice [197]. The}

inconsistent results between these studies using n-3 PUFA diets may arise from differences in IBD induction models, intervention times (prevention vs. treatment), the dose of PUFA, lipid profiles of diets, or changes in bioavailability. Furthermore, Trebble et al. [198] demonstrated that the production of the pro-inflammatory cytokines TNF and IL6 by PBMCs appeared to show a “U-shaped” dose-response after n-3 PUFA supplementation. In this study, the supplementation of dietary fish oil in healthy humans resulted in decreased TNF and IL6 production by LPS-stimulated PBMCs at the lowest level (0.3 g/d n-3 PUFA). Maximum inhibition was observed at intermediate levels (1.0 g/d n-3 PUFA), and the least inhibition was seen at the highest supplementation levels (2.0 g/d n-3 PUFA). It was hypothesised that molecular mechanisms by which n-3 PUFA affects cytokine production could have maximum effects at different intake levels of n-3 PUFA, resulting in the observed “U-shaped” dose-response curve [198]. The timing of PUFA supplementation may also be an important factor [199]; feeding diets before colitis induction could have different effects (preventive) when compared with a therapeutic approach (where diets are fed when colitis is already present [199]).

Figure 1.6 Metabolism of omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFA) from precursor fatty acids with focus on arachidonic acid (AA) in the n-6 pathway and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the n-3 pathway [adapted from 200].

The anti-inflammatory effects of n-3 PUFA may be different when in a food due to changes in bioavailability of n-3 PUFA [201]. Bioavailability is defined as “the proportion of a drug or nutrient which enters the circulation when introduced into the body and so is able to have an active effect” [202]. Fish intake may increase the bioavailability of n-3 PUFA because (i) the ingestion of whole foods is followed by a more effective activation of digestion/absorption in the intestine compared to capsules [203-205]; (ii) lipids in salmon are mostly in the form of triacylglycerol (TG), with n-3 PUFA mostly in central position of the TG molecule (which facilitates absorption [206, 207]); and (iii) the bioavailability of EPA is improved when ingested with a high-fat meal [208]. Human studies have reported that eating salmon was more efficient in increasing n-3 PUFA levels in serum and plasma than taking fish oil capsules [204, 205]. However, this contrasts to results from Arterburn et al. [209], who reported that algal oil capsules and cooked salmon were comparable in terms of bioavailability of DHA to plasma phospholipids and erythrocytes. The results of these studies [204, 205, 209] may depend on several factors [210], including genetic differences of the individual subjects, the oxidation rate of n-3 PUFA in capsules, and differences in encapsulation (e.g. hard vs. soft gelatine capsules).

The duodenum and jejunum are the main sites for lipid digestion [211, 212], where digestive enzymes hydrolyse TG (the main form of dietary lipids) to monoacylglycerols and fatty acids and further form mixed micelles (Figure 1.7). Micelles diffuse to the brush border of the enterocytes, where the lipid substances are absorbed by epithelial cells. Absorption is facilitated by transport proteins, namely plasma membrane- associated fatty acid binding protein (FABPpm) [213], CD36 [214] and fatty acid transport protein 4 (FATP4) [215]. Whether passive diffusion or transporter-facilitated absorption dominates is unclear, and while this may depend on fatty acid concentration, Schwenk et al. [216] suggested transporter-mediated absorption dominates. In the enterocytes, fatty acids and 2-monoacylglycerols are transported to the endoplasmic reticulum in association with a family of proteins known as cytoplasmic fatty acid binding proteins (FABPc) [as reviewed in 217] where they are re-esterified, forming first diacylglycerols, then TG (Figure 1.7). Newly synthesised TG are transported out of the enterocyte in the form of chylomicrons and enter the lymphatic capillaries in the intestinal villi. Chylomicrons are supplied to peripheral tissues via the bloodstream, while only approximately 2% of dietary lipids enter the colon via the intestinal lumen [218].

Figure 1.7 Intestinal digestion and absorption of dietary lipids. (A) The main site for lipid absorption is the small intestine, where large fat droplets are emulsified with bile salts from the liver. Pancreatic juices hydrolyse TG to monoacylglycerol, diacylglycerol, and fatty acids, and phospholipids to lysophosphatidic acid. Micelles are formed from digestive products and mixed with bile salts and cholesterol. (B) Mixed micelles diffuse to the brush-border of the enterocytes, where micelles are broken down and digestive products enter the enterocytes by passive diffusion and facilitated by transporters. Intracellular enzymes re- esterify free fatty acids and monoacylglycerols sequentially to diacylglycerols and TG. TG enter the lymphatic capillaries in form of chylomicrons. Adapted from Shi et al. [219] with permission from

Macmillan Publishers Ltd: Nature Reviews.

BS: Bile salts; CL: Cholesterol; CM: Chylomicrons; DAG: Diacylglycerol; DGAT: Diacylglycerol acyltransferase; FA: Free fatty acids; LPA: Lysophosphatidic acid; MAG: Monoacylglycerol; MGAT: Monoacylglycerol acyltransferase; MTP: Microsomal triglyceride transfer protein; PL: Phospholipids; TG: Triacylglycerol.