Influence of fatty acids composition in different tissue of mice feeds with fish oils

Omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) play an important role in human health. Fish oils enriched with EPA and DHA have commercialized in triacylglycerol (TAG) and ethyl ester forms (EE). In this study, we compared the impact of diets containing fish oil in ethyl ester and triacylglycerol forms as a lipid source in five different tissues as liver, skeleteral muscle, brain, and epididymal white adipose tissue (WAT). The DHA levels were higher in the WAT and skeletal muscle of TAG and EE groups in comparison with the SB group. The body weight and brain, liver, epididymal WAT, and gastrocnemius muscle weights, and serum glucose, TG, cholesterol were not different between the groups. Thus, Research, Society and Development, v. 10, n. 16, e338101623706, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.23706 2 we conclude that EPA and DHA in the form of EE or TAG influence the fatty acids composition of different tissues.


Introduction
The fatty acids (FA) are distributed in all the tissues of living organisms. They have been received special attention considering that the quantity and quality of FAs consumed in the diet play different roles in physiological processes (Wanten & Calder, 2007). Nowadays, the demand for food products enriched with omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and omega-6 long-chain polyunsaturated fatty acids (n-6 LC-PUFA) have increased. It is due their beneficial effects on human health. In especial, n-3 LC-PUFA modulates cell membrane permeability and reduces thrombosis, cardiovascular diseases, diabetes, inflammatory diseases, and neurological disorders (Wang et al., 2018). Two important n-3 LC-PUFA are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These n-3 LC-PUFAs show beneficial effects in patients with hyperlipidemia, diabetes, cancers, inflammation, and neurodegenerative diseases (Dave & Routray, 2018). EPA and DHA may be obtained from marine animals (Zhang, Xu, Wang, & Xue, 2019) or from α-linolenic acid (LNA) in a biossintetic process with the participation of the elongases and desaturases. Interestingly, LNA and linoleic acid (LA) are not produced in the human body. However, they be obtained from the diet (Liu et al., 2020).
Fish oil enriched with EPA and DHA can be considered a functional food because it has positive effects on health (Sarojnalini & Hei, 2019). It is one of the most commonly prescribed supplements. The consumption of marine fish oil rich in n-3 LC-PUFA content used as a food supplement has stimulated the production in the pharmaceutical industry (Viswanathan, Verma, Ganesan, & Manivannan, 2017). The EPA and DHA are generally commercialized as triacylglycerol ester (TAG), or ethyl ester (EE). After FAs production, the preparation of fish oil supplements by the industries occurs from two different pathways. The first step, the produced FAs reacts with enzymes to bond the FA into the glycerol molecule again (reesterification process) which results in a new TAG molecule. In the second one, the EE formed results by the reaction between FA and ethyl alcohol. The n-3 PUFA fish oils are found in the TAG form (MacKay, 2007).
These different chemical structures of EPA and DHA may produce differences in the bioavailability of n-3 PUFA in the bloodstream (MacKay, 2007). Ester form of FA is not usually found in the human diet. Thus, is necessary remove the ethyl group and convert the FA back into a TAG molecule during absorption (Bezard, Blond, Bernard, & Clouet, 1994). Previous studies suggests that the uptake of free FA occurs after the hydrolysis of their esters by esterases in the intestinal lumen. Some reports have shown that EE molecular forms of FA EPA and DHA may affect absorption, bioavailability, storage, and activity in the body (Lindblom et al., 2018). However, the absorption of different forms of EPA and DHA acids is still not clear.
In the liver, the DHA controls lipid metabolism stimulating β-oxidation or decreasing the FAs synthesis through the downregulation of fatty acid-synthase (Nakamura, Yudell, & Loor, 2014). In the skeletal muscle, n-3 PUFA improve muscle anabolism in addition to reduce protein degradation (Jeromson, Gallagher, Galloway, & Hamilton, 2015). Other study suggests that DHA regulates epididymal WAT metabolism managing pro and anti-inflammatory actions. It alleviates adipocytes abnormalities (Bargut, Santos, Machado, Aguila, & Mandarim-de-Lacerda, 2017). In brain tissue, n-3 PUFAs improve the fluidity of neuronal membranes, acting in pivotal functions, such as the release of neurotransmitters (Meng et al., 2010). Thus, the uptake of different chemical forms of n-3 LC-PUFAs contained in fish oil supplements must be investigated. In this study, EPA and DHA in two different forms in diet were evaluated regarding the liver, brain, muscle, and epididymal WAT tissues FA composition through the incorporation of TAG and EE forms in the diet of mice. Diets composition were based on the purified diet for rodents proposed by the American Institute of Nutrition (AIN-93-M) (Reeves, Nielsen, & Fahey Jr, 1993) with modifications Silva-Santi et al. (2016), as described in Table 1. SB: diet containing lard and soybean oil as a lipid source; EE: diet containing swine lard and fish oil in the form of EE as a lipid source; TAG: diet containing lard and fish oil in the form of TAG; n-6: omega 6; n-3: omega 3. Source: Authors.

Animals and diets
Six After a three-day acclimation, the mice were randomly divided into 3 groups: The control group received a diet containing lard and soybean oil as a lipid source (SB group). The EE group received swine lard and fish oil in the form of EE as a lipid source (EE group). The TAG group received lard and fish oil in the form of TAG as a lipid source (Table 1). With this procedure it was possible to determine which forms of TAG or EE, incorporated in the diet is capable of influencing the composition of FA in liver, brain, muscle, serum and epididymal WAT.
The diets were available in feeders of PVC tubes coated with metal ring to prevent material degradation. The feeder was fixed at the box, allowing only the access of the animal's head to its interior, so that the diet is not spread and allowing a more exact quantification of its consumption.
The animals were submitted to the treatment period of 56 days. Food intake was assessed daily. After the respective treatment period, the animals were weighed and fasted 15 h before euthanasia. The mice were euthanized and the livers, brains, muscles, and epididymal WAT were collected, quickly frozen in liquid nitrogen, and stored in a freezer at -80 °C. Serums from all animals was also collected and was used for analysis of glucose, triglycerides and cholesterol.

Direct Derivatization of FA
Livers, brains, skeletal muscles and epididymal WAT were crushed still frozen, under liquid nitrogen, in a gral with the aid of pistil until obtaining homogeneous powder. The direct derivatization of FA was performed according to the method developed by Figueiredo et al. (2016). About 100 mg of each sample was weighed into 10 cm high test tubes. Then 2 mL of NaOH (1.5 mol L -1 in methanol) was added to the tubes and the contents were macerated with a glass stick to increase the contact surface. Then the test tubes were placed in an ultrasonic bath, Eco-Sonics Q 5.9/25 (Unique, São Paulo, Brazil) with 165 W of power at 25 kHz for 5 min at 30 ºC. Then 2 mL of H2SO4 (1.5 mol L -1 in methanol) was added and the tubes were again brought to the ultrasonic bath under the above conditions. Then, 1 mL of n-heptane was added to the tubes which were then stirred for 30 s and centrifuged at 2000 rpm for 1 min. Finally, 500 μL of internal standard (23:0 methyl ester) was added to the tubes and the supernatant was collected and transferred to amber flasks for further chromatographic analysis. This procedure was performed for each sample in triplicate. This methodology only derives the free FAs and the TAGs present in the samples. In this way, the FAs constituting other biomolecules are not capable of being derivatized.

FA Composition by Gas Chromatography with Flame Ionization Detector (GC-FID)
FA methyl esters (FAMEs) were separated and quantified on a Thermo Scientific gas chromatograph, trace ultra 3300 model, equipped with a flame ionization detector, a split/splitless injector and a CP-7420 fused silica capillary column (Select FAME, 100 m long, 0.25 mm internal diameter and 0.25 μm fine cyanopropyl film as the stationary phase). The operating parameters were: column temperature 165 °C for 18 min and then heated to 235 °C (4 °C min -1 ) for 20 min; the injector and detector temperatures were kept constant at 230 and 250 °C, respectively; the gas flows were 1.2 mL min -1 for the entrainment gas (H2), 30 mL min -1 for the gas make up (N2), and in the flame ionization detector, 30 and 300 mL min -1 of H2 gas and synthetic air, respectively; the samples were injected in the split mode at 1:40, in which the injection volume was 1 μL.
The FAMEs were identified by comparison to the retention times of the sample constituents with Sigma FAMEs.
Correction factors were used to obtain the concentration values of FAs according to and the amount of FA were calculated in mg 100 g -1 of sample.
Absolute quantification of the FAMEs was performed by internal standardization using standard methyl ester of tricosanoic acid (23:0 methyl ester) (Sigma, St. Louis, USA). The percentages were determined by integrating the peak areas with the ChromQuest 5.0 software.

Statistics Analysis
The results were submitted to analysis of variance (ANOVA), and the means were compared by Tukey's test with a significance level of 95% using R: A language and environment for statistical computing (R Development Core Team 3.0.1.,

2013).
In the fatty acid composition data, monounsaturated fatty acids, saturated fatty acids, sum of omega-6 and 3 fatty acids, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, SB, TAG and ethyl ester of the liver, epididymal wat and brain samples, were submitted to a multivariate exploration technique, the chosen technique was the principal component analysis (PCA), being performed in the Rstudio software version 1.4.1106.

Results and Discussion
In this study, we compared the impact of diets containing: a) lard and soybean oil as a lipid source (SB diet), b) lard and fish oil in the form of ethyl ester (EE diet) as a lipid source, c) lard and fish oil in the form of TAG (TAG diet) as a lipid source.
It is shown the FA composition of SB diet, EE diet, and TAG diet at  TAG: diet containing lard and fish oil in the form of TAG; MUFAmonounsaturated fatty acids; SFA -Saturated fatty acids; n-6sum of omega-6 fatty acids; n-3sum of omega-3 fatty acids; AAarachidonic acid; EPAeicosapentaenoic acid; DHAdocosahexaenoic acid. Source: Authors.
Initial body weight, body weight gain, brain weight, liver weight, epididymal WAT weight, gastrocnemius muscle weight, blood glucose, triacylglycerol, and cholesterol serum levels were not different btween the groups SB, TAG and EE (Table 3). Table 3. Initial body weight, body weight gain, gastrocnemius muscle, liver, brain, epididymal WAT weights (g) and blood levels of glucose (mg/dl), triacylglycerol (mg/dl), cholesterol (mg/dl). Liu et al. (2020) evaluated the effect of different diets on body weight and fat mass in mice for 12 weeks and observed a slight change in the body weight gain for low n-3:n-6 ratio and SFA compared to medium and high n-3:n-6 ratios.

SB
Furthermore, the glucose, cholesterol, and triacylglycerol levels have increase with the decrease of n-3/n-6 ratios (Liu et al., 2020). Similar results have reported before with replacement from a high SFA diet to a n-3 PUFA to evaluate glucose intolerance and vascular dysfunction (Lamping et al., 2013). On the order hand just enrichment with n-3 PUFA diet did not exhibit a difference in the same parameters (Bowman et al., 2018).
Moreover, the high values of n-3 PUFA into the TAG diet imply better results to SFA:n-3, myristic acid:DHA, and n-6:n-3 ratios in comparison with other treatments. The TAG diet showed the lower n-6:n-3 ratio. The balanced n-6:n-3 ratio (1-2/1) is associated with the prevention of cardiac diseases and obesity. Current diets in different cultures worldwide showed values ranging from 38-50 (Urban India); 16.74 (US); 15 (UK and northern Europe); 4 (Japan). Furthermore, the lower hepatic SFA: n-3 and n-6: n-3 ratios in TAG and EE groups liver could indicate a better anti-inflammatory state. In contrast, high SFA values is associated with inflammation (Simopoulos, 2016).
The tetracosanoic acid (24:0) was detected only in the SB group. The 24:0 content could be related to successive reactions of elongation and saturation. Three successive elongation reactions from oleic acid are necessary to produce that acid whose the last two steps are catalyzed by elongase 1 (Kihara, 2012).
The TAG diet exhibited higher content of arachidic acid (20:0) and gondoic acid (20:1n-9). These results are associated with the activity of elongases (Kihara, 2012). The very-long-chain composition of SFA such as arachidic acid (20:0) decreases the risk of diseases, such as diabetes. On the other hand, the composition of very-long-chain MUFAs as gondoic acid appears to decrease cardiovascular disease risk (Yang, Emma-Okon, & Remaley, 2016).
The liver plays an important role in FA metabolism (Rui, 2014), as the synthesizes of DHA and AA from their precursors. The conversion of linoleic (n-6 series) and α-linolenic (n-3 series) acids, respectively in DHA and AA consists of a succession of desaturation and elongation steps (Kihara, 2012). In this context, the AA is a substrate for inflammatory eicosanoids synthesis. Nevertheless, the n-3 PUFA is related to anti-inflammatory eicosanoids synthesis. Higher content of n-3 may contribute to the anti-inflammatory state in tissues of TAG (Wanten & Calder, 2007). Also, EPA and DHA are substrates for resolvins and protectins, which are powerful anti-inflammatory molecules (Calder, 2015). In skeletal muscle (Table 5), the content of 24:0, 24:1n-9, 20:4n-6, and 20:3n-3 were higher (p<0.05) in the SB diet when compared with TAG and EE groups. In muscle, the higher amount of tetracosanoic is related to higher inflammation Research, Society and Development, v. 10, n. 16, e338101623706, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.23706 (Antunes et al., 2020). There was no statistical difference among the myristic, palmitic,stearic,docosanoic,arachidic,palmitoleic,vaccenic,oleic,gondoic,linoleic,and EPA content between all the diets. On the other hand, the incorporation of DHA was over 100% (p<0.05) higher in the TAG and EE groups in comparison with the control group. Jeromson, Gallagher, Galloway, & Hamilton (2015) reported that at least 2 weeks of a new diet are enough to promote significant change in its lipid composition and showed that skeletal muscle is responsive to the diet changes. Brown et al. (2019) have exhibited that n-3 LC-PUFA (EPA and DHA) displayed beneficial effects on skeletal muscle protein turnover. And the anti-inflammatory response played by n-3 PUFA can be a key role in the preservation of lean muscle mass in post-operative cancer patients (Ryan et al., 2009).
The total SFA was higher (p<0.05) in the TAG group than in SB and EE treatments. Total MUFA and n-6 were lower (p<0.05) in the EE group, compared to the others. The sum of n-3 was lower (p<0.05) in the SB group compared to TAG and EE. The SFA/n-3 and n-6/n-3 ratios were higher (p<0.05) in the SB in comparison with TAG and EE groups. In this way, the n-3 enriched diet contributed to decreasing the n-6/n-3 ratio wich can have beneficial effects in the body. The western diet has a high n-6/n-3 ratio of 20:1; High content of n-6 PUFA cause an unbalanced ratio since the lack of n-3 PUFA. The unbalance among n-3 and n-6 PUFA may exacerbate a pro-inflammatory state (Jeromson, Gallagher, Galloway, & Hamilton, 2015).
Rocha-Rodrigues et al. (2017) evaluated the impact of physical exercise on FA of the WAT in response to a high-fat diet regimen. They found the same FA quantified in this study ( Table 6). The SB diet showed higher (p<0.05) content of eicosadienoic acid than TAG and EE treatments. However, in the WAT the EPA and DHA had higher amounts (p<0.05) in TAG and EE groups in comparison with the SB diet. Earlier study reported that the ability to storage DHA is limited in concerning with brain, liver, and muscle. This can be related with the substantial escape of DHA, liberated by lipoprotein lipase from WAT to the blood (Arterburn, Hall, & Oken, 2006).
The TAG and EE groups showed lower 11-14-eicosadienoic acid in epididymal WAT. Also, the diets showed no difference in the total SFA, MUFA, PUFA, n-6, and n-3. However, SFA:n-3 and n-6:n-3 ratios were higher (p<0.05) in the EE than in SB and TAG groups.
The brain has a FA composition tightly regulated and exhibits lower response to diet composition changes in comparison with liver, skeletal muscle, and heart (Silva-Santi et al., 2018). In addition, the brain displays a preference for DHA uptake concerning the other FAs. Not always brain DHA levels are dependent on dietary n-3 PUFA. The dietary n-3 PUFA deficiency has more impact in some specific situations as pregnancy and lactation (Levant, Ozias, & Carlson, 2006).
In general, the changes observed in FAs of the liver, muscle and epididymal WAT tissues the sum of all the FA evaluated, SFA, MUFA, and PUFA were similar between the groups. SFA is associated with the development of metabolic dysfunction. On the other hand, some MUFAs, and PUFAs have been linked to the positive effects on metabolic function (Jeromson, Gallagher, Galloway, & Hamilton, 2015). Higher n-3 PUFA content of TAG and EE diets were not able to limit TG deposition in the liver, as reported by Di Minno et al. (2012) moreover, the TAG and EE groups presented decreased in some n-6 PUFA as AA either in liver and muscle and 11-14-eicosadienoic acid in epididymal WAT. These results are in agreement with other studies that showed decreased AA contents with DHA supplementation (Arterburn, Hall, & Oken, 2006).
To the best view of the data, the principal component analysis (PCA) has been applied. The variables evaluated were AA, DHA, EPA, n-3 PUFA, n-6 PUFA, MUFA, and SFA in three treatments (SB, TAG, and EE). We choose two components to explain all data (Figure 1 A). The first component (PC1) explained 86.86% of the variance. The second component (PC2) explained 12.93%. Thus, the sum of PC1 and PC2 results at 99.79% of the total data variance. Part B of Figure 1 shows the proximity of all the samples forming two distinct groups. The liver, muscle, and epididymal specimens generated the first group. The second group was composed by brain samples. The MUFA and SFA variables are responsible for the formation of two groups due to the higher negative eigenvalues. The higher positive eigenvalues were the EPA, DHA, AA, and n-3 variables. The biggest difference found among the results obtained for the brain is because the FA composition of the brain is less sensitive to diet changes (Silva-Santi et al., 2018).

Conclusion
For the first time, we showed the influence in the fatty acid composition of liver, brain, skeletal muscle, and epididymal WAT in mouse´s model fed with different chemical structures of n-3 long-chain fatty acids like EPA and DHA.
The increase of DHA content in the skeletal muscle and epididymal WAT compared to the control diet proves the LC-PUFA n-3 accumulation. Furthermore, the performance animal parameters did not display a difference among all the samples.
Therefore, EPA and DHA in the form of EE or TAG influence the FA composition in muscle, liver, and epididymal WAT, but not in the brain. The authors suggest that further studies should be carried out with other animal species, including fish included in the diet of the present study, since it influenced the fatty acid composition of the tissues of mice and could positively affect health.