Laboratoire de Biochimie-Nutrition Humaine, Agrocampus Rennes, INRA USC 2012, Rennes, France

Transcription

1 Animal (2008), 2:4, pp & The Animal Consortium 2008 doi: /S animal Substitution of dietary oleic acid for myristic acid increases the tissue storage of a-linolenic acid and the concentration of docosahexaenoic acid in the brain, red blood cells and plasma in the rat V. Rioux -, D. Catheline, E. Beauchamp, J. Le Bloc h, F. Pédrono and P. Legrand Laboratoire de Biochimie-Nutrition Humaine, Agrocampus Rennes, INRA USC 2012, Rennes, France (Received 10 May 2007; Accepted 6 December 2007) Various strategies have been developed to increase the cellular level of (n-3) polyunsaturated fatty acids in animals and humans. In the present study, we investigated the effect of dietary myristic acid, which represents 9% to 12% of fatty acids in milk fat, on the storage of a-linolenic acid and its conversion to highly unsaturated (n-3) fatty acid derivatives. Five isocaloric diets were designed, containing equal amounts of a-linolenic acid (1.3% of dietary fatty acids, i.e. 0.3% of dietary energy) and linoleic acid (7.0% of fatty acids, i.e. 1.5% of energy). Myristic acid was supplied from traces to high levels (0%, 5%, 10%, 20% and 30% of fatty acids, i.e. 0% to 6.6% of energy). To keep the intake of total fat and other saturated fatty acids constant, substitution was made with decreasing levels of oleic acid (76.1% to 35.5% of fatty acids, i.e. 16.7% to 7.8% of energy) that is considered to be neutral in lipid metabolism. After 8 weeks, results on physiological parameters showed that total cholesterol and low-density lipoprotein-cholesterol did not differ in the diets containing 0%, 5% and 10% myristic acid, but were significantly higher in the diet containing 30% myristic acid. In all the tissues, a significant increasing effect of the substitution of oleic acid for myristic acid was shown on the level of both a-linolenic and linoleic acids. Compared with the rats fed the diet containing no myristic acid, docosahexaenoic acid significantly increased in the brain and red blood cells of the rats fed the diet with 30% myristic acid and in the plasma of the rats fed the diet with 20% myristic acid. Arachidonic acid also increased in the brain of the rats fed the diet with 30% myristic acid. By measuring D6-desaturase activity, we found a significant increase in the liver of the rats fed the diet containing 10% of myristic acid but no effect at higher levels of myristic acid. These results suggest that an increase in dietary myristic acid may contribute in increasing significantly the tissue storage of a-linolenic acid and the overall bioavailability of (n-3) polyunsaturated fatty acids in the brain, red blood cells and plasma, and that mechanisms other than the single D6-desaturase activity are involved in this effect. Keywords: dietary myristic acid, (n-3) polyunsaturated fatty acid biosynthesis and metabolism, rat, D6-desaturase Introduction It is well known that a cellular increase in (n-3) polyunsaturated fatty acids (PUFA), and especially in highly unsaturated derivatives such as docosahexaenoic acid (DHA; C22:6 n-3) is beneficial for human health, especially during early neural tissue development (Salem et al., 2001; Alessandri et al., 2003) and for the prevention of cardiovascular diseases (Harris and von Schacky, 2004; Harper et al., 2006). In many countries, however, the intakes of both a-linolenic acid (ALA; C18:3 n-3) and DHA (Burdge - Vincent.Rioux@agrocampus-rennes.fr and Calder, 2005; Denomme et al., 2005) are lower than the current dietary recommendations (Simopoulos et al., 1999). Various dietary strategies have been proposed to increase their cellular level: (i) consumption of oils rich in ALA (Harper et al., 2006), but the rate of in vivo conversion of ALA to highly unsaturated fatty acids (FA) is low (Burdge and Calder, 2005; Hussein et al., 2005), and its effective conversion to DHA in mammalian tissues, although recently quantified in the plasma of rats as 10-fold higher than brain consumption rates (Igarashi et al., 2007), is controversial (Barcelo-Coblijn et al., 2005; Lin and Salem, 2005), because of the competition of multiple substrates for D6-desaturase (D Andrea et al., 2002); (ii) consumption 636

2 Effect of dietary myristic acid on (n-3) PUFA of fish, fish oil or microalgae oil (Geppert et al., 2005) directly providing the long-chain (n-3) derivatives; and (iii) consumption of both ALA and long-chain (n-3) derivatives, by using, for example, products from breeding animals that have been fed with extruded linseeds (Weill et al., 2002). It is therefore important to define the factors that can regulate the conversion of ALA to DHA. Among these factors, we and others have recently suggested that myristic acid (MY; C14:0), which represents 9% to 12% of FA in milk fat, may be an activator: (i) in cultured rat hepatocytes, we have shown that MY increases D6-desaturase activity in a dose-dependent manner (Jan et al., 2004); (ii) in vivo, when MY was supplied for 2 months in the diet of rats (from 0.2% to 1.2% of dietary energy), a dose response accumulation of eicosapentaenoic acid (EPA; C20:5 n-3) and eicosatrienoic acid (C20:3 n-6) to a lesser extent, but no significant effect on DHA, was shown in the liver and plasma (Rioux et al., 2005); and (iii) in an interventional study in humans (Dabadie et al., 2005), a diet containing 1.2% of MY from dairy fat significantly enhanced EPA and DHA levels in the plasma phospholipids (PL), and DHA level in the plasma cholesteryl esters (CE), compared with a diet containing 0.6% of MY. More recently, in the same population (Dabadie et al., 2006), when the intake of MY increased from 1.2% to 1.8% of energy, EPA and DHA decreased significantly in the plasma PL, and EPA also decreased in the CE. This last result suggests that, in humans, the potential beneficial effect of MY on the circulating (n-3) PUFA profile is highest around 1.2% of total dietary energy. Concerning other physiological parameters, MY is known to have negative effects on cholesterol metabolism (Hayes and Koshla, 1992; Salter et al., 1998), when provided at high level in the diet (more than 4% of energy). The first goal of the present study was to investigate further the putative beneficial effect of various doses of dietary MY on (n-3) PUFA cellular levels and hepatic D6-desaturase activity in the rat. As a secondary goal, the investigation was also used to reevaluate the threshold level of dietary MY leading to negative dysfunction of cholesterol metabolism. Diets were designed to contain myristic acid from traces (0.02% of FA) to intermediate levels (5% to 10% of FA) similar to MY in bovine and human milk (Jensen et al., 1990), then to doses exceeding the typical intake range (20% to 30% of FA). In order to keep the other saturated fatty acids (palmitic and stearic acids) and total fat intake constant, substitution was made with decreasing levels of oleic acid (OL; C18:1 n-9) that is considered to be neutral in lipid metabolism (Blankenhorn et al., 1990; Truswell and Choudhury, 1998). Diets were carefully controlled with respect to essential FA content, to contain strictly equal amounts of ALA (1.3% of FA) and linoleic acid (LA; C18:2 n-6: 7.0% of FA), i.e. 0.3% and 1.5% of energy, respectively, and to produce a constant dietary ratio of 5, that follows the recommended current human nutritional guidelines. Materials and methods Chemicals Solvents and chemicals were obtained from Fisher (Elancourt, France) or Sigma (St-Quentin Fallavier, France). [1-14 C]-a-linolenic acid was from Perkin Elmer Life Sciences (Le Blanc Mesnil, France) and [1-14 C]-eicosatrienoic acid was from American Radiolabeled Chemicals (Isobio, Fleurus, Belgium). Kits for plasma cholesterol and triacylglycerol (TG) were purchased from Bio-mérieux (Lyon, France). Fractionated butter fat was provided by Lactalis (Retiers, France). Tripalmitin was from Sigma. Trimyristin was extracted from powdered nutmeg by refluxing with diethyl ether (250 ml/100 g of nutmeg) for 5 h. After filtration and concentration, the solution was cooled down to 08C. Trimyristin was collected by filtration, washed twice with cold ethanol and recrystallized from acetone. Diets Diets were prepared at the Unité de Production d Aliments Expérimentaux (UPAE, INRA, Jouy en Josas, France). They were isocaloric and varied only in the type of fat used, in order to contain increasing amounts of myristic acid. The composition was as follows (g/100 g): 42.6 corn starch, 19.8 casein, 21.3 sucrose, 0.9 cellulose, 3.6 mineral mix, 0.9 vitamin mix, 0.9 agar-agar and 10.0 lipid. Fat provided 22% of total energy. Lipid fractions of the diets were made by mixing the following fat: olive oil, rapeseed oil, corn oil, fractionated butter fat containing myristic acid mainly in sn-2 position in the TG, tripalmitin and trimyristin (Table 1). The diets, named MY0, MY5, MY10, MY20 and MY30, contained, therefore, increasing amounts of MY, decreasing amount of OL and constant amounts of essential LA and ALA. Cholesterol in the diets was (g/100 g): 0.00, 0.06, 0.07, 0.05 and 0.03 for MY0, MY5, MY10, MY20 and MY30, respectively. Animals Animals were cared in accordance with the National Institute of Health Guide. Sprague-Dawley male rats (60 g body weight, 6 week old at the beginning of the experiment) were obtained from the breeding center R. Janvier (Le Genest-St-Isle, France) and maintained on rat chow (nutriment A04, Scientific Animal Food and Engineering, Augy, France) with free access to water for 1 week before the study. Then, the rats were randomly assigned to five groups (n 5 6 per group) and fed ad libitum with the five diets described above (MY0 to MY30) for 8 weeks. At the end of this period, the rats were fasted overnight. On the following morning, they were anaesthetized with an intraperitoneal injection of pentobarbital (7.5 mg/100 g body weight) (Rioux et al., 2005). Blood samples were drawn into heparinated tubes by cardiac puncture. The liver, brain and epidydimal fats were removed, weighed, snap-frozen in liquid nitrogen and stored at 2808C. The plasma was separated from the blood cells by centrifugation ( g, 637

3 Rioux, Catheline, Beauchamp, Le Bloc h, Pédrono and Legrand Table 1 Fat mixture and fatty acid (FA) composition (wt%) of the five experimental diets Diets MY0 MY5 MY10 MY20 MY30 Fat Olive oil Rapeseed oil Corn oil Butter fat Trimyristin Tripalmitin FA C4: C6: C8: C10: C12: C14:0 myristic acid C16: C18: SSFA C14:1 n C16:1 n C18:1 n-9 oleic acid C18:1 n Trans MUFA SMUFA C18:2 n-6 linoleic acid C20:3 n C20:4 n Sn-6 PUFA C18:3 n-3 a-linolenic cid Sn-3 PUFA C18:2/C18:3 ratio MY 5 myristic acid; MUFA 5 monounsaturated fatty acid; PUFA 5 polyunsaturated fatty acid. 10 min, 48C). Plasma cholesterol and TG were assayed with commercial enzymatic kits, according to the manufacturer s instructions. Lipid extraction and FA analysis Lipids from liver (3 g) and epidydimal adipose tissue (100 mg) were extracted using a mixture of dimetoxymethane/methanol (4 : 1 v/v) (Guillou et al., 2002). Lipids from plasma and red blood cells (RBC) were extracted from 1 ml of samples with a mixture of hexane/isopropanol (3 : 2 v/v), after acidification with 1 ml HCl 3 mol/l (Rioux et al., 2000). Total lipid extracts from liver, red blood cells, plasma, adipose tissue and brain were saponified for 30 min at 708C with1mlof 0.5mol/lNaOHinmethanolandmethylatedwith1mlBF 3 (14% in methanol) for 15 min at 708C. FA methyl esters were extracted twice with pentane and analyzed by GC using an Agilent Technologies 6890N (Bios Analytic, Toulouse, France) with a split injector (40 : 1) at 2508C and a bonded silica capillary column (30 m mm; BPX 70; SGE, Villeneuve-St-Georges, France) with a polar stationary phase of 70% cyanopropylpolysilphenylene-siloxane (0.25 mm film thickness). Helium was used as carrier gas (average velocity 24 cm/s). The column temperature program started at 1508C, ramped at 28C/min to 2208C and held at 2208C for 10 min. The flame ionization detector temperature was 2508C. Desaturase assays Fresh liver samples (3 g) were homogenized in 12 ml of 50 mmol/l phosphate buffer (ph 7.4) containing 0.25 mol/l sucrose, and centrifuged twice at g for 30 min. The resulting post-mitochondrial supernatant was diluted three times and used for D6- and D5-desaturase assays (Rioux et al., 2005). Enzymatic activity was determined using a 1-ml assay mixture containing 100 ml of supernatant (600 to 800 mg protein), 150 mmol/l phosphate buffer (ph 7.16), 6 mmol/l MgCl 2, 7.2 mmol/l ATP, 0.54 mmol/l CoA and 0.8 mmol/l NADH. The incubation was carried out at 378C for 20 min, after addition of 60 nmol of [1-14 C]-a-linolenic acid or [1-14 C]-eicosatrienoic acid (740 MBq/mmol) for D6- or D5-desaturase, respectively. The reactions were stopped by adding 1 ml of 2 mol/l KOH in ethanol. After 30 min at 708C, the FA were liberated by acidification, extracted with diethylether, converted to FA naphthacyl esters (Rioux et al., 1999) and separated on high-performance liquid chromatography (Alliance 2695 integrated system, Waters, St Quentin en Yvelines, France). Peaks corresponding to radiolabeled FA substrate and product of each desaturase assay were collected and subjected to liquid scintillation counting (Packard Tri-Carb 1600 TR, Meriden, CT, USA). The enzyme activity was determined and expressed as pmol substrate converted to product per min per mg of protein. Protein in the supernatant used for the desaturase assays was determined by a modified Lowry procedure. Results expression and statistical analysis The results are expressed as mean 6 SD (n 5 6). The data of the five dietary groups were analyzed by using the S-PLUS statistical software (Insightful, Seattle, WA, USA). Statistical differences between means were determined by ANOVA and two-tailed Student s t-test. A value of P, 0.05 was considered to be statistically significant. Results Effect of the diets on the physiological parameters Rats were fed the five diets (Table 1) for 8 weeks. The dietary consumption was identical for the five groups. At the end of this period, body weights, tissue weights, plasma lipid concentrations and total fatty acid content were analyzed in six rats for each group (Table 2). Animals from the MY30 and MY20 groups were significantly heavier than the other three groups, like their liver weights. For the five groups, the total FA content in the liver, plasma, brain and adipose tissue was similar (Table 2). Total cholesterol was not statistically different between the MY0, MY5 and MY10 638

4 Effect of dietary myristic acid on (n-3) PUFA Table 2 Physiological parameters measured in the rats fed the five experimental diets for 8 weeks Physiological parameter MY0 MY5 MY10 MY20 MY30 Weight Body (g) a a a b b Liver (g) a a a b b Brain (g) a a a a a Plasma lipids Total cholesterol (mg/ml) a a,b a,b b,c c HDL-cholesterol (mg/ml) a a a a a Total cholesterol/hdl-cholesterol a a a,b b b LDL-Cholesterol (mg/ml) a a a,b a,b b TG (mg/ml) a a,b a,b b b Total FA Liver (mg/g) a a a a a Plasma (mg/ml) a a a a a Adipose tissue (mg/g) a a a a a Brain (mg/g) a a a a a MY 5 myristic acid; HDL 5 high-density lipoprotein; LDL 5 low-density lipoprotein; TG 5 triglycerides. Values on the same line with different superscript letters are significantly different (P, 0.05). LDL-cholesterol was calculated with the following formula: LDL-cholesterol 5 (total cholesterol) 2 (HDL-cholesterol) 2 (TG/5). groups and significantly higher in the MY30 animals, with intermediate level in the MY20 group. High-density lipoprotein (HDL)-cholesterol did not differ significantly between diets, and changes in plasma cholesterol levels occurred mainly in low-density lipoproteins (LDL) (MY30 group compared withmy0-my10 groups). Finally, a significant increase in the circulating TG levels was shown in rats from the MY20 and MY30 groups as compared with the MY0 group, with intermediate values in the MY5 and MY10 groups. Effect of the diets on the tissue levels of C14:0 and C18:1 n-9 The diets were experimentally designed (Table 1) to contain increasing amounts of MY (from 0% to 30% of FA) and decreasing levels of OL (from 76.1% to 35.5%). We first analyzed the effect of these diets on the tissue levels of both MY and OL (Tables 3 7). In every tissue (RBC, plasma, liver, brain and adipose tissue), a clear dose response enrichment of MY was shown, that follows the level of MY in the diets (Tables 3 7). The increase was especially high in adipose tissue (from 1% to 14%; Table 5) and especially low in the brain, but still significant (from 0.1% to 0.2%; Table 7). On the contrary, except in adipose tissue, which clearly reflected the decreasing dietary levels of OL, only a moderate decrease of OL was shown in RBC, plasma and brain (Tables 3, 4, 7). No effect of the diets was shown in the liver on OL levels (Table 6). Effect of the diets on the tissue storage of C18:3 n-3 and C18:2 n-6 The five diets were designed to contain strictly equal amounts of ALA (1.3% of FA) and LA (7.0%) and no other highly unsaturated FA of the (n-3) and (n-6) series, except for traces of AA (Table 1). After 8 weeks of the diets, the level of ALA significantly increased as a function of dietary MY in every tissue (Tables 3 6), except in the brain in which it was not detected (Table 7). When analyzing LA levels, a similar increasing relationship with the MY dietary level was shown in every tissue (Tables 3 7). Effect of the diets on the tissue level of (n-3) derivatives Compared with the rats fed the diet containing no MY, a significant increase in DHA and total (n-3) PUFA was shown in the brain (Table 7) and RBC (Table 3) of the rats fed the diet with 30% MY and in the plasma (Table 4) of the rats fed the diet with 20% MY. No effect on DHA was shown in the liver (Table 5). Effect of the diets on the tissue level of (n-6) derivatives Compared with the rats fed the diet containing no MY, a significant increase in AA and total (n-6) PUFA was shown only in the brain (Table 7). No other significant effect was shown in total (n-6) PUFA derivatives in RBC (Table 3), plasma (Table 4) and liver (Table 6). Effect of the diets on D6- and D5-desaturase activities We then tested whether the different cellular levels of MY could have an effect on hepatic D6- and D5-desaturase activities, as already shown in cultured rat hepatocytes for D6-desaturase (Jan et al., 2004). Figure 1a shows that, in the MY10 group, D6-desaturase activity (using C18:3 n-3 as the substrate) was significantly higher than that in the MY0 group and also than that in the MY20 and MY30 groups. No significant effect of the diets was shown on hepatic D5-desaturase activities with C20:3 n-6 as the substrate (Figure 1b). 639

5 Rioux, Catheline, Beauchamp, Le Bloc h, Pédrono and Legrand Table 3 Fatty acid (FA) composition (% of total FA) of red blood cells (RBC) total lipids C14: a b c d c,d C16: a a a a a C18: a a a a a SSFA a a a a a C16:1 n a b a,b b b C16:1 n a a a a a C18:1 n a a,b a,b a,b b C18:1 n a a a a a SMUFA a a a a a C18:2 n a a,b b,c c b,c C20:2 n a b b,c a,b,c c C20:3 n a a,b b a,b a,b C20:4 n a a a a a C22:4 n a a a a a C22:5 n a a a a a S n-6 w/o C18: a a a a a S n-6 PUFA a a a a a C18:3 n a,b a b,c c c C20:5 n a b b a b C22:5 n a b a,b b,c c C22:6 n a a,b a,b a,b b S n-3 w/o C18: a b b,c b,c c S n-3 PUFA a b b,c b,c c MY 5 myristic acid; SFA 5 saturated fatty acid; MUFA 5 monounsaturated fatty acid; PUFA 5 polyunsaturated fatty acid. Values on the same line with different superscript letters are significantly different (P, 0.05). Discussion A number of parameters have been shown to regulate the tissue storage of ALA and the efficiency of its conversion to DHA (Burdge and Calder, 2005) in humans and animals: the physiological or pathological status, the amount of dietary ALA, the LA/ALA ratio in the diet, the dietary background and the level of other FA in the diet. Within last parameter, this study was aimed at investigating the effect of dietary myristic acid on the storage of ALA, the biosynthesis of highly unsaturated (n-3) derivatives from the precursor and the overall bioavailability of these PUFA. Isocaloric diets containing increasing amounts of myristic acid (from 0% to 30% of total FA) and decreasing amounts of oleic acid (from 76% to 35%) were designed (Table 1) and given to rats for 8 weeks, as already done in previous studies investigating the effect of dietary myristic acid in rats (Rioux et al., 2005) and humans (Dabadie et al., 2005 and 2006). The present study shows first that plasma cholesterol and TG levels were statistically similar in animals from the MY0, MY5 and MY10 groups (Table 2). Higher doses of myristic acid (20% and 30% of FA, i.e. 4.4% and 6.6% of dietary energy) had a significant negative effect on cholesterol metabolism and particularly on the total cholesterol : HDL-cholesterol ratio, which is used as a predictor of coronary heart disease risk in humans. Epidemiological and clinical studies have shown that myristic acid consistently increases animal and human blood cholesterol concentrations more than other FA when given at high doses, exceeding 4% of dietary energy (Hayes and Koshla, 1992; Salter et al., 1998). From these studies, it has therefore been suggested that myristic acid may be the most hypercholesterolemic saturated FA, followed by lauric acid (C12:0) and palmitic acid (C16:0). More recently, nutritional studies analyzing the effect of physiological doses (1.0% to 2.5% of dietary energy) of myristic acid have shown no effect on plasma total cholesterol in rats (Rioux et al., 2005), or a non-significant increase that reflected an increase in HDL-cholesterol but not LDLcholesterol in hamsters (Loison et al., 2002). As recently described in healthy men and women (Tholstrup, 2006), our results suggest that a moderate consumption of dairy products does not increase the risk of cardiovascular diseases (Dabadie et al., 2005; Tholstrup et al., 2003). In this study, we also hypothesized that dietary myristic acid could influence the storage of ALA and the biosynthesis of highly unsaturated (n-3) derivatives from the precursor. In order to compare the five groups, the diets were designed to contain only the n-6 and n-3 precursors (ALA and LA) and not the derivatives, in a ratio that meets human essential FA requirements (Simopoulos et al., 1999). The results first show an increasing accumulation of both ALA and LA as a function of increasing level of MY in the diet, in all the tissues analyzed (Tables 3 7). From this result, one can hypothesize that, depending on the diet, either both essential FA have been less b-oxidized or less 640

6 Effect of dietary myristic acid on (n-3) PUFA Table 4 Fatty acid (FA) composition (% of total FA) of plasma total lipids C12: a a a a a C14: a b c d e C16: a a,b b,c b,c c C18: a a,b a,b a,b b SSFA a a,b b,c b,c c C16:1 n a a a a a C16:1 n a b b b b C18:1 n a b b,c b,c c C18:1 n a a a a a SMUFA a b a,b,c b,c c C18:2 n a a,b b b c C20:2 n a a a a a C20:3 n a a,b a,b b b C20:4 n a a a a a C22:4 n a a a,b b,c c C22:5 n a a a,b b b S n-6 w/o C18: a a a a a S n-6 PUFA a a a a a C18:3 n a a,b a,b a,b b C20:5 n a a,b a,b a,b b C22:5 n a a a b b C22:6 n a,b a a b a,b S n-3 w/o C18: a a a b b S n-3 PUFA a a a b b MY 5 myristic acid; SFA 5 saturated fatty acid; MUFA 5 monounsaturated fatty acid; PUFA 5 polyunsaturated fatty acid. Values on the same line with different roman superscripts are significantly different (P, 0.05). Table 5 Fatty acid (FA) composition (% of total FA) of adipose tissue total lipids C12: a b c b d C14: a b c d e C16: a b,c b b,c c C17: a b b c d C18: a b b a c SSFA a b c d e C16:1 n a a b c d C16:1 n a b b b b C18:1 n a b b c d C18:1 n a b a,b a a,b SMUFA a b c d e C18:2 n a b,c b c,d d C20:2 n a a,b b b b S n-6 PUFA a b b b,c c C18:3 n a b b c c S n-3 PUFA a b b c c MY 5 myristic acid; SFA 5 saturated fatty acid; MUFA 5 monounsaturated fatty acid; PUFA 5 polyunsaturated fatty acid. Values on the same line with different superscript letters are significantly different (P, 0.05). converted to longer unsaturated derivatives by elongation and desaturation. The b-oxidation pathway is described as a major catabolic utilization of both ALA and LA, since a large proportion of ALA (60% to 85%) and similar levels of LA goes through this pathway (Cunnane and Anderson, 1997). MY is also known to be a good substrate for this catabolic pathway (Rioux et al., 2000). Therefore, the presence of increasing doses of myristic acid and lower levels of oleic acid in the diets may have led to an increased substrate competition with ALA and LA for the b-oxidation 641

7 Rioux, Catheline, Beauchamp, Le Bloc h, Pédrono and Legrand Table 6 Fatty acid (FA) composition (% of total FA) of liver total lipids C14: a b c d d C16: a b b,c c c C18: a a a b a,b SSFA a b c b,c c C16:1 n a a a a C16:1 n a b b b b C18:1 n a a a a a C18:1 n a a a a a SMUFA a a a a a C18:2 n a b b a,b b C20:2 n a b a,b c c C20:3 n a,b a,b a b a,b C20:4 n a a a a a C22:4 n a a a a a C22:5 n a a a a a S n-6 w/o C18: a a a a a S n-6 PUFA a a a a a C18:3 n a a,b a,b b b C20:5 n a,b a a b b C22:5 n a a a a a C22:6 n a a a a a S n-3 w/o C18: a a a a a S n-3 PUFA a a a a a MY 5 myristic acid; SFA 5 saturated fatty acid; MUFA 5 monounsaturated fatty acid; PUFA 5 polyunsaturated fatty acid. Values on the same line with different roman superscripts are significantly different (P, 0.05). Table 7 Fatty acid (FA) composition (% of total FA) of brain total lipids C14: a a b c b,c C16: a a,b a,b a,b b C18: a a a,b a b C20: a a a a a SSFA a a a,b a b C16:1 n a a a a a C16:1 n a a b b b C18:1 n a a a a b C18:1 n a a a a a C20:1 n a a a a a SMUFA a a a a a C18:2 n a a,b b,c b c C20:2 n a a a a a C20:3 n a a,b b a,b b C20:4 n a a a,b a b C22:4 n a a a,b a b C22:5 n a a a a a S n-6 w/o C18: a a a,b a b S n-6 PUFA a a a,b a,b b C18:3 n-3 ND ND ND ND ND C20:5 n a a a a a C22:5 n a b b b b C22:6 n a a a,b a,b b S n-3 w/o C18: a a a,b a,b b S n-3 PUFA a a a,b a,b b MY 5 myristic acid; SFA 5 saturated fatty acid; MUFA 5 monounsaturated fatty acid; PUFA 5 polyunsaturated fatty acid; ND 5 not detected. Values on the same line with different superscript letters are significantly different (P, 0.05). 642

8 Effect of dietary myristic acid on (n-3) PUFA Figure 1 Effect of increasing doses of myristic acid from 0% (MY0) to 30% (MY30) on (a) D6-desaturase and (b) D5-desaturase activities (mean 6 SD, n 5 6). In vitro desaturase activities were measured as described in materials and methods. Significant differences (P, 0.05) between diets are indicated by differing letters. pathway, explaining the increased storage of these two FA. In addition, as opposed to OL, a significant amount of MY was probably absorbed via the portal vein, going directly to the liver where it was b-oxidized. This competition is probably dependent on the ratio of both MY and OL dietary levels, since in the first study we made on rats fed MY at 1.2% of dietary energy, ALA content decreased in adipose tissue (Rioux et al., 2005). In that previous study, we chose to keep dietary OL level constant (54% of FA) and to decrease stearic acid to compensate for the increase in MY. Concerning the (n-3) highly unsaturated derivatives, although none of them was present in the diets, experimental results showed a significant accumulation of DHA and total (n-3) derivatives as a function of dietary MY in the brain (Table 7), plasma (Table 4) and RBC (Table 3), but not in the liver (Table 6). These results are in accordance with those obtained in an interventional study in humans (Dabadie et al., 2005), showing that a moderate intake (up to 1.2% of total energy) of myristic acid during 5 weeks was associated with a significant increase in EPA and DHA in the plasma CE fraction. In the rat, when MY was supplied for 2 months in the diet (from 0.2% to 1.2% of dietary energy), a dose response accumulation of EPA, and eicosatrienoic acid (C20:3 n-6) to a lesser extent, but no significant effect on DHA was shown in the liver and plasma (Rioux et al., 2005). In this previous study, the level of dietary MY was low compared to the present study, and only the liver and plasma were analyzed. To decrypt the mechanisms underlying the increasing tissue accumulation of DHA and (n-3) derivatives, we measured desaturase activities involved in the conversion of ALA to DHA. The results showed that hepatic D6-desaturase activity was significantly increased from MY0 to MY10, and then went back to baseline from MY10 to MY30 (Figure 1). The first part of this result is similar to that obtained in cultured rat hepatocytes, showing that myristic acid, supplied in the culture medium, increased D6-desaturase activity in a dose-dependent manner (Jan et al., 2004). When considering the decreasing level of dietary oleic acid, which is a substrate of D6-desaturase, hypothesis can also be made that it could have an increasing effect on D6-desaturase activity measured with ALA as substrate. However, the present study shows that decreasing the dietary level of oleic acid had no effect on its own hepatic cellular level (Table 6), whereas myristic acid was increased in the liver. Moreover, this putative effect of OL on D6-desaturase has not been shown in other nutritional studies (Mahfouz et al., 1984). When higher doses of myristic acid (MY20 and MY30) were given to the rats, hepatic D6-desaturase activity came back to baseline levels (Figure 1). Considering that D6-desaturase is a membrane enzyme, this last effect can be explained by changes of biophysical properties of the membranes containing higher levels of myristic acid in MY20 and MY30 rats. Altogether, in this study, the significant increase in D6-desaturase activity (Figure 1) did not correspond to a significant increase in the tissue content of DHA (Tables 3, 4 and 7). On the contrary, at higher dietary amounts of MY, no change in desaturase activity was noted, whereas an increase in DHA content was reported. To explain this gap, several hypotheses may be formulated. First, the shift in time between desaturase activity measurement and FA storage in the tissues may be considered. Second, ALA may be the limited source of longer chain (n-3) PUFA in mammals. Thus, desaturase activities, like elongase activities, may not be at maximal values in the tissues, depending on the availability of each substrate in the pathway of DHA biosynthesis from ALA. Third, hypothesis can also be made that long-chain (n-3) FA derivatives may either have been synthesized directly in the brain and red blood cells, or synthesized in the liver and subsequently distributed to other tissues, which could explain the decreasing level of DHA in the liver, and the tissue-dependent effect of these results (Tables 3 7). For long, ALA has been thought to be predominantly desaturated and elongated in the liver, with subsequent distribution of the converted products to other tissues through the plasma. When the diet contains DHA, ALA did not contribute appreciably to DHA within brain phospholipids of adult rats (Demar et al., 2005). However, some recent results have suggested that DHA could be synthesized directly in the brain from ALA (Barcelo-Coblijn et al., 2005). We cannot confirm these results, since we were not able to measure desaturase activities in the brain (data not shown). One way to resolve the gap between fatty acid composition and desaturase and elongase 643

9 Rioux, Catheline, Beauchamp, Le Bloc h, Pédrono and Legrand activities might be to infuse appropriate labeled fatty acid into blood, and to measure rates of oxidation or elongation/ desaturation directly in the liver, as recently published (Igarashi et al., 2007). In conclusion, these results suggest that increasing longchain (n-3) PUFA dietary intake is not the exclusive way to increase long-chain (n-3) PUFA cellular levels. Although the conversion of ALA to DHA is low, the presence of a nutrient like MY can help to reach this goal by modulating both the level of cellular ALA and its conversion to DHA. From a nutritional point of view, myristic acid should therefore be considered for its potential beneficial effect on (n-3) FA cellular availability, although we keep in mind its atherogenic effect when given in excess. Acknowledgments The authors thank ARILAIT RECHERCHES (France) for constructive scientific discussion and financial support. We are grateful to Dr Le Ruyet (Lactalis) for providing the fractionated butter fat, X. Blanc (UPAE, INRA, Jouy en Josas, France) for the preparation of the diets, M. Bouriel and K.L. Cung for able technical assistance and animal care, and A. Delvaux for editorial assistance. References Alessandri JM, Poumès-Ballihaut C, Langelier B, Perruchot MH, Raguénez G, Lavialle M and Guesnet P Incorporation of docosahexaenoic acid into nerve membrane phospholipids: bridging the gap between animals and cultured cells. American Journal of Clinical Nutrition 78, Barcelo-Coblijn G, Collison LW, Jolly CA and Murphy EJ Dietary a-linolenic acid increases brain but not heart and liver docosahexaenoic acid. Lipids 40, Blankenhorn DH, Johnson RL, Mack WJ, el Zein HA and Vailas LI The influence of diet on the appearance of new lesions in human coronary arteries. Journal of the American Medical Association 263, Burdge GC and Calder PC Conversion of a-linolenic acid to longer-chain polyunsaturated FAs in human adults. Reproduction Nutrition Development 45, Cunnane SC and Anderson MJ The majority of dietary linoleate in growing rats is b-oxidized or stored in visceral fat. Journal of Nutrition 127, Dabadie H, Peuchant E, Bernard M, LeRuyet P and Mendy F Moderate intake of myristic acid in sn-2 position has beneficial lipidic effects and enhances DHA of cholesteryl esters in an interventional study. Journal of Nutritional Biochemistry 16, Dabadie H, Motta C, Peuchant E, LeRuyet P and Mendy F Variations in daily intakes of myristic and a-linolenic acids in sn-2 position modify lipid profile and red blood cell membrane fluidity. British Journal of Nutrition 96, D Andrea S, Guillou H, Jan S, Catheline D, Thibault JN, Bouriel M, Rioux V and Legrand P The same rat D6-desaturase not only acts on 18- but also on 24-carbon FAs in very-long-chain polyunsaturated FA biosynthesis. Biochemical Journal 364, Demar Jr JC, Ma K, Chang L, Bell JM and Rapoport SI a-linolenic acid does not contribute appreciably to docosahexaenoic acid within brain phospholipids of adult rats fed a diet enriched in docosahexaenoic acid. Journal of Neurochemistry 94, Denomme J, Stark KD and Holub BJ Directly quantitated dietary (n-3) FA intakes of pregnant Canadian women are lower than current dietary recommendations. Journal of Nutrition 135, Geppert J, Kraft V, Demmelmair H and Koletzko B Docosahexaenoic acid supplementation in vegetarians effectively increases omega-3 index: a randomized trial. Lipids 40, Guillou H, Martin P, Jan S, D Andréa S, Roulet A, Catheline D, Rioux V, Pineau T and Legrand P Comparative effect of fenofibrate on hepatic desaturases in wild-type and peroxisome proliferator-activated receptor a-deficient mice. Lipids 37, Harper CR, Edwards MJ, DeFilippis AP and Jacobson TA Flaxseed oil increases the plasma concentrations of cardioprotective (n-3) fatty acids in humans. Journal of Nutrition 136, Harris WS and von Schacky C The omega-3 index: a new risk factor for death from coronary heart disease? Preventive Medicine 39, Hayes KC and Koshla P Dietary FA thresholds and cholesterolemia. FASEB Journal 6, Hussein N, Ah-Sing E, Wilkinson P, Leach C, Griffin BA and Millward DJ Long-chain conversion of [ 13 C]linoleic acid and a-linolenic acid in response to marked changes in their dietary intake in men. Journal of Lipid Research 46, Igarashi M, DeMar Jr JC, Ma K, Chang L, Bell JM and Rapoport SI Upregulated liver conversion of a-linolenic acid to docosahexaenoic acid in rats on a 15 week n-3 PUFA-deficient diet. Journal of Lipid Research 48, Jan S, Guillou H, D Andréa S, Daval S, Bouriel M, Rioux V and Legrand P Myristic acid increases D6-desaturase activity in cultured rat hepatocytes. Reproduction Nutrition Development 44, Jensen RG, Ferris AM, Lammi-Keefe CJ and Henderson RA Lipids of bovine and human milks: a comparison. Journal of Dairy Science 73, Lin YH and Salem N In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method. Journal of Lipid Research 46, Loison C, Mendy F, Sérougne C and Lutton C Dietary myristic acid modifies the HDL-cholesterol concentration and liver scavenger receptor BI expression in the hamster. British Journal of Nutrition 87, Mahfouz MM, Smith TL and Kummerow FA Effect of dietary fats on desaturase activities and the biosynthesis of FAs in rat liver microsomes. Lipids 19, Rioux V, Catheline D, Bouriel M and Legrand P High performance liquid chromatography of fatty acids as naphthacyl derivatives. Analusis 27, Rioux V, Lemarchal P and Legrand P Myristic acid, unlike palmitic acid, is rapidly metabolized in cultured rat hepatocytes. Journal of Nutritional Biochemistry 11, Rioux V, Catheline D, Bouriel M and Legrand P Dietary myristic acid at physiologically relevant levels increases the tissue content of C20:5 n-3 and C20:3 n-6 in the rat. Reproduction Nutrition Development 45, Salem N, Litman B, Kim H and Gawrisch K Mechanisms of action of docosahexaenoic acid in the nervous system. Lipids 36, Salter AM, Mangiapane EH, Bennett AJ, Bruce JS, Billett MA, Anderton KL, Marenah CB, Lawson N and White DA The effect of different dietary fatty acids on lipoprotein metabolism: concentration effects of diets enriched in oleic, myristic, palmitic and stearic acids. British Journal of Nutrition 79, Simopoulos AP, Leaf A and Salem N Essentiality of and recommended dietary intakes for omega-6 and omega-3 fatty acids. Annals of Nutrition and Metabolism 43, Tholstrup T Dairy products and cardiovascular disease. Current Opinion in Lipidology 17, Tholstrup T, Vessby B and Sandström B Difference in effect of myristic and stearic acid on plasma HDL cholesterol within 24 h in young men. European Journal of Clinical Nutrition 57, Truswell AS and Choudhury N Monounsaturated oils do not all have the same effect on plasma cholesterol. European Journal of Clinical Nutrition 52, Weill P, Schmitt B, Chesneau G, Daniel N, Safraou F and Legrand P Effect of introducing linseed in livestock diet on blood fatty acid composition of consumers of animal products. Annals of Nutrition and Metabolism 46,