Forty-eight male Wistar rats (Charles River Laboratories, baseline body weight 75–100 g) were fed a standard diet (A04, SAFE Diets, France). The animals were acclimated 1 week before being distributed into the four experimental groups (Fig. 1). They were housed in individual cages under conditions of constant temperature, humidity, and a standard light-dark cycle (12 h/12 h). Tap water and standard diet were provided ad libitum.

In experiment 1 (Exp. 1), the rats (n=12/group) received daily, in addition to the standard diet, either control pellets (CP) with control oil (CO) or WA pellets with CO. The CO was palm oil which is free from alpha-linolenic acid (ALA), the precursor of VLC n-3 fatty acids, and served as an isoenergetic control matrix (Fig. 1).

In experiment 2 (Exp. 2), the rats (n=12/group) received daily, in addition to the standard diet, either CP with linseed oil (LO) or WA pellets with LO. LO was used to provide ALA (18:3n-3) (Fig. 1).

Both CO and LO were provided by gavage and brought 9 kcal/g. The amount of fat provided by the oils was adjusted to the recommendations of the American Institute of Nutrition Rodent Diets-93 (12). Thus, the quantity of linseed oil used for gavage was 200 µL/day corresponding to 92 mg/day of ALA. The WA pellets were prepared by mixing 2 g of the WA layer flour containing 26.7 mg/g dry weight of total FA with 2.5 mL water and 12.5 mg commercial white sugar. The aleurone fraction was provided by Barilla G. e R. F.lli, Parma (Italy), and obtained as follows: durum wheat (Triticum turgidum L. subsp. Durum) kernels went through three subsequent steps of debranning. The material obtained was micronized and separated by a turbo separator into inner and outer parts of the aleurone layer. The outer part of the aleurone, characterized by small particles size (340–400 nm), was used for this study.

The WA pellets provided, in addition to the standard diet, a total of 53.4 mg of FA per day and per rat. The CP were prepared by mixing 1 g of standard A04 ground in flour with 2.5 mL water and 12.5 mg refined commercial white sugar. Mass density of 1 g of the flour of standard A04 was equivalent to 2 g of the WA layer flour. The CP provided, in addition to the standard diet, a total of 8.4 mg of FA per day and per rat. The WA and CP were prepared daily. Table 1 shows the lipid composition of the WA and A04 used in the studies.

In all the experiments body weights and food consumption were recorded weekly. At the end of Exp. 1 and 2, blood was collected on fasted rats and plasma was stored at −80°C. Immediately after blood collection, the rats were euthanized and their liver sampled, weighed, frozen in liquid nitrogen, and stored at −80°C until analysis. Finally, the urinary concentration of phenolic acids and their metabolites was estimated in the urine samples collected overnight during the last week of treatment of the animals in Exp. 1.

Plasma and liver lipids were extracted in hexane/isopropanol as described (5–7). Briefly, methylated fatty acids were extracted with hexane, separated, and quantified by GC using a 6850 Series gas chromatograph system (Agilent Technologies, Palo Alto, CA, USA). Methyl ester peaks were identified by comparing their retention time to those of a standard mixture. Saturated, mono-, and poly-unsaturated fatty acid levels were expressed as a percentage of total fatty acids. Total cholesterol and triglycerides were measured using standard methodology on a Synchron Clinical System LX20 (Beckman Coulter, Brea, CA, USA).

Phenolic acids in CP and WA, as well as phenolic metabolites in rat urine, were analyzed using an Accela UHPLC 1250 equipped with a linear ion-trap-mass spectrometer (LTQ XL, Thermo Fisher Scientific Inc., San Jose, CA, USA) fitted with a heated-electrospray ionization probe (H-ESI-II; Thermo Fisher Scientific Inc.). Separations were performed using a BlueOrchid-1.8 C18 column (50×2 mm) (Knauer, Berlin, Germany). Pure helium gas (99.999%) was used for collision-induced dissociation (CID). The HPLC-MSⁿ conditions used for the detection are available in the Supplementary file.

For the extraction of the free phenolic fraction, 500 mg of sample were mixed with 6 mL of water. The sample was mixed for 2 min, left under agitation for 30 min at room temperature, and centrifuged at 9,200×g for 10 min. Finally, the supernatant was recovered and stored at −20°C until the UHPLC-MSⁿ analysis. For the extraction of the bound phenolic fraction, the extraction residue was further digested with 1.5 mL of 2 mol/L sodium hydroxide at room temperature for 1 h. After alkaline hydrolysis, the pH of the mixture was adjusted to 3 by adding 1.35 mL of 3 mol/L citric acid. The samples were extracted with 6 mL of ethyl acetate and treated as the water extract. The ethyl acetate extracts were evaporated to dryness and the residue was dissolved in methanol. Bound fraction extracts were kept at −20°C in the dark prior to UHPLC-MSⁿ analysis. The HPLC-MSⁿ conditions used for the detection are available in the Supplementary file.

Urine samples of rats treated with CP or WA were filtered through a 0.45 µm nylon filter before UHPLC-MSⁿ analysis. The HPLC-MSⁿ conditions used for the detection are available in the Supplementary file.

Statistics were realized using the software Minitab version 15 (GrimmerSoft, Paris, France). The difference between each fatty acid was compared with unpaired Student's t-test. The difference between each phenolic acid metabolites concentration excreted in urine after the consumption of the WP or CP were compared by non-parametric Mann–Whitney test.

In Exp. 1, body weight after 12 weeks (373±26 g vs. 367±13 g) and food intake (23.8±2.6 g/day vs. 22.1±1.4 g/day) were not different in the two groups. In Exp. 2, body weight after 12 weeks (376±23 g vs. 378±21 g) and food intake (24.1±1.0 g/day vs. 23.3±1.3 g/day) were not different in the two groups.

Several phenolic acids have been identified and quantified in the WA and CP pellets. The sum of phenolic acids was 28 mg/g for WA and 10 mg/g for CP, mostly in bound form (Table 2). FA was by far the most abundant compound as monomeric and dimeric forms.

Several phenolic acid metabolites have been identified in the urines of rats receiving either CP or WA (Table 3). Six metabolites of FA were present in conjugated forms (as sulfate and glucuronide derivatives). The rats fed WA excreted more FA-O-sulfate compared to controls (p<0.05) whereas no significant difference was found for the other major metabolites of other phenolic acids, notably hippuric acid and enterolactone.

In Exp. 1 with CO, cholesterol and triglycerides were not different in the two groups (Table 4). Regarding plasma fatty acids, only EPA was significantly higher among rats receiving WA compared with CP (Table 4). In Exp. 2 with LO; cholesterol, triglycerides, and fatty acid composition were not different in the two groups (Table 4). Liver fatty acids were not different in rats receiving WA or CP, regardless of which oil used for the treatment (data not shown).