1. Introduction
It is known that polyunsaturated fatty acids of the n-3 series (n-3 PUFA) demonstrably reduce the plasma levels of total cholesterol and triacylglycerols (TAG) in humans [
1,
2]. Both n-3 and n-6 PUFA play an important role in the formation, development, modulation, and stopping of inflammatory processes.
N-3 PUFA include α-linolenic acid (ALA), which is essential for humans; however, ALA received in the diet can be desaturated and elongated to long-chain PUFAs (LC-PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic (DHA). The effectiveness of converting 18-carbon ALA to 20-carbon EPA in healthy young men is approximately 8% [
3,
4]. This conversion is higher in women and corresponds to 21% [
4]. Most studies summarized in the article of Domenichiello et al. [
5] demonstrate that less than 1% of ALA is converted to DHA. It thus follows that direct consumption of EPA and DHA is the most effective means for increasing the content of health-giving n-3 LC-PUFA in the lipids of the blood plasma and phospholipids of the cell membranes.
Metabolic syndrome is associated with obesity, dyslipidemia, hypertension, and impaired glucose tolerance, and is thus closely connected with type 2 diabetes (T2D). Replacing saturated fatty acids (SFA) by mono-unsaturated FA (MUFA) can demonstrably reduce the risk of development of metabolic syndrome [
6]. Especially n-3 PUFA have the greatest ability to reduce the TAG level and total serum cholesterol (S-chol), increase the level of serum high-density lipoprotein cholesterol (S-HDLC) and activate the lipid metabolism [
6,
7]. Impaired glucose tolerance, as a risk factor of insulin resistance, is one of the connecting links and simultaneously the first stage of T2D. According to the meta-analysis of Qian et al. [
8], PUFAs lead to a reduction of fasting glycemia by 0.87 mmol/l. Also, the effects of n-3 PUFAs on platelet function have been studied intensively [
9,
10]. Platelet aggregation assays are performed with agents that physiologically activate platelets in vivo, for example, adenosine diphosphate (ADP), arachidonic acid, collagen, and epinephrine. The results of clinical studies of the effect of n-3 PUFA on platelet aggregation are ambiguous. Meta-analysis of nine studies [
10] demonstrated that daily supplementation with n-3 PUFAs significantly reduces ADP-induced platelet aggregation compared with a placebo with a trend towards a decrease in collagen-induced aggregation, but not in arachidonic acid-induced aggregation.
ALA occurs primarily in flaxseed oil with about 40% content of ALA and, to a lesser degree, in canola oil with a 10% content, as well as in soya oil and walnuts. The main sources of EPA and DHA are saltwater fish [
11,
12,
13,
14], krill [
15], and microalgae [
16,
17,
18]. Cardoso et al. give the amount of DHA present in Western food as approximately 100 mg/day [
11]. At the same time, the recommended value for consumption of EPA and DHA for the European adult population is in the range 250–500 mg/day [
11,
12,
19,
20]. Consequently, in addition to food supplements containing fish oil, other means are sought to supplement the n-3 LC-PUFA content in our diets. The smell of fish oil containing approximately 18% EPA and 12% DHA, or its concentrate [
21], makes it quite unacceptable for many individuals.
Another possibility represents stearidonic acid (SDA; C18:4n3), from which EPA is synthesized more effectively than from ALA. Prasad et al. mentions 14–16% conversion of SDA to EPA [
22] and Bowen et al. even 33% [
23]. A high amount of SDA is reported in hemp oil, blackcurrant oil, and echium oil, however its conversion to DHA is low [
22]. Another option lies in the breeding of farm animals fed a mixture containing ALA derived from natural plant sources, such as flaxseed and flaxseed oil, where it is assumed that ALA is converted to higher n-3 LC-PUFA in these animals. Cortinas et al. showed a 16-fold increase in ALA in chicken breast meat to a level of 4.1 g/kg and an almost 30-fold increase in ALA in thigh meat with skin to 31.4 g/kg, when applied approximately 7% flaxseed oil and 2% fish oil into feed mixture [
24]. The increase in EPA and DHA, derived from the fish oil or from the ALA conversion, reached 2.39 g/kg (340% increase) for EPA and 1.13 g/kg (66% increase) for DHA; 0.3 g/kg EPA (130% increase) and 0.4 g/kg EPA (40% increase) were measured in the chicken breast. The greater part of the FA received is thus stored in the skin and subcutaneous fat. Zuidhof et al. documented triple levels of ALA and EPA in the meat of broilers using a feed mixture containing 10% flaxseed, while DHA level was unchanged [
25]. The enrichment of eggs with n-3 PUFAs using various oilseeds and fish oil is summarized in a review by Fraeye et al. [
26]. The 2% addition of flaxseed oil in the feed mixture reached 15 times the original level of ALA (14.88 mg/g of yolk), EPA increased from zero level to 0.37 mg/g of egg yolk, and DHA is more than doubled to 6.49 mg/g of yolk [
27]. In another study, Benavides reported an 11.8-fold increase in ALA in eggs using 10% flaxseed in the feed mixture [
28]. Some algae that directly produce LC-PUFA can also be used to prepare feed mixtures [
16,
17,
18,
29,
30]. In this way products such as chicken and eggs enhanced in n-3 FA can be obtained directly from these animals. Disadvantages lie in the lower organoleptic quality and stability of the obtained products against spontaneous oxidation [
20], which is suppressed by the addition of antioxidants, such as vitamin E to the feed mixture. Pork [
31], beef [
32], and carp meat [
33] with elevated contents of n-3 FA can be obtained similarly.
The increased demand for enriched products led to the idea of developing basic commonly consumed foods that would contain naturally higher contents of PUFA and LC-PUFA, i.e., chicken and eggs, hereafter designated in this article as “omega-3 meat” and “omega-3 eggs”. In the Czech Republic, eggs are considered a basic food with an annual consumption of 14.6 kg brutto (263 eggs) per person in 2018; the annual consumption of chicken in the Czech Republic is 28.4 kg per person according to the Czech Statistical Office [
34]. RABBIT Trhový Štěpánov Inc. (Trhový Štěpánov, Czech Republic) produces omega meat and eggs by adding flaxseed with high ALA content directly to the feed mixtures of chickens and laying hens. The submitted study is part of an extensive project concerned with the development of feed mixtures, the production of omega-3 chickens and eggs, and their wide distribution to the retail network in the Czech Republic. Because of their great nutrient value, the successful production of omega-3 meat and eggs would represent a major potential that would find its place on the market even if the retail prices of food were to increase. Here we deal with the aspect of the effect of eight-week consumption of omega-3 eggs and meat on selected biochemical and hematological blood parameters, body composition measured by the bio-impedance method, and the complete spectrum of fatty acids (FA) in the blood plasma and in the phospholipids of the erythrocyte membrane. The blood plasma lipids provide information on the actual lipid metabolism, while the membrane phospholipids provide long-term information on lipid consumption and metabolism.
2. Materials and Methods
2.1. Production of Omega-3 Meat and Eggs
RABBIT Trhový Štěpánov Inc. provided for the production of omega-3 chicken and eggs. Enrichment of foodstuffs in n-3 PUFA is based on the addition of flaxseed oil, as a natural source of n-3 PUFA, to the feed mixtures. The addition of flaxseed oil was optimized and finally an amount of 2% wt. flaxseed oil was added to the mixture for feeding chickens to produce omega-3 meat and 1% wt. flaxseed oil was added to the mixture for feeding laying hens for production of omega-3 eggs. The control groups were fed the standard feed mixture, in which soya is the source of lipids, once again in an amount of 2% wt. for the production of meat and 1% wt. for the production of eggs. Further addition of flaxseed oil to the feed mixture causes diarrhea in chickens and laying hens and is unfeasible from a feed point of view. The meat and eggs were analyzed for their contents of n-3 and n-6 PUFA at the Department of Food Analysis and Nutrition of the University of Chemistry and Technology in Prague using gas chromatography (GC) technique (
Table 1).
The addition of flaxseed oil to the feed mixture was manifested in an increase in LC-PUFA from 19.9 mg to 53.8 mg per 100 g of meat (170% increase), and from 70 mg to 110 mg per 100 g of egg (57% increase). The overall amount of n-3 PUFA in the meat was changed from 250 mg to 900 mg in 100 g of meat (260% increase) and from 110 mg to 190 mg per 100 g of whole egg (73% increase). The increase in n-6 PUFA in the meat and eggs is substantially less and the thus-produced foodstuffs can be designated as omega-3 meat and eggs.
2.2. Dietary Study
This was a randomized study and subjects were blinded to the n-3 FA intervention. A total of 28 healthy 18–25-year-old men, students of Charles University, Third Faculty of Medicine, were recruited into the study. The volunteers were divided into two groups of 14 individuals each, and further designated as the control group (n = 14) and the omega-3 group (n = 14). Participants did not suffer from any chronic condition or take any medication, didn’t undergo any restrictive diet and did not engage in any extremely intense physical activity (defined as more than 12 h per week). Basic anamnestic data, nutritional habits, and lifestyle behaviour were self-recorded by participants in a questionnaire. All the participants were informed in detail about the study and signed a written consent. The study was approved by the Ethical Committee of the Third Faculty of Medicine (Charles University, Prague, Czech Republic), head of review board Dr. Marek Vácha, PhD. All participants were informed about the study and planned examinations and signed informed consent.
The set of volunteers was randomly divided into two groups; one received an experimental diet containing the enriched eggs and chicken and the other received a control diet. The experimental and control diets were administered four times a week in the regime of one egg (57 g netto) and 120 g of meat per serving. The control diet was prepared and administered in the same way as the experimental diet, with the difference that ordinary unenriched products (chicken and eggs) were used. According to the plan, the nutrition intervention lasted eight weeks. The meals were planned by a registered dietitian, prepared in the canteen of the Faculty Hospital Královské Vinohrady and provided to the participants under the supervision of a responsible member of the experimental team.
The average daily intake of n-3 PUFA from enriched chicken meat and eggs during the intervention is summarized in
Table 2. Due to the fact that the total dietary intake of LC-PUFA was not monitored, the obtained values are related to the recommended daily intake of LC-PUFA in the Western European diet, which is 250–500 mg/day. The performed intervention represents a total daily increase of LC-PUFA intake by 37 mg compare to the control group, which is 7-15% of the recommended daily dose. The design of the food intervention was deliberately set to correspond to the usual eating habits in the population and a further increase in the consumption of eggs and meat over four eggs and four servings of meat per week) seems unrealistic in long-term practice.
The baseline diets of participants were not determined, but all the participants were instructed not to change their lifestyle, especially their dietary habits and physical activity throughout the duration of the study and not to undertake any restrictive dietary regime.
2.3. Anthropometric and Body Composition Data
The basic anthropometric data (weight, height, Body Mass Index (BMI)) were determined at the beginning and end of the intervention, the body composition (by the bio-impedance method) and the blood pressure were measured. Body composition values were measured by Body composition analyzer (Tanita MC 180 MA, Amsterdam, The Netherlands). All the participants were instructed not to change their lifestyle, especially their dietary habits and physical activity throughout the duration of the study and not to undertake any restrictive dietary regime. In case of acute illness, the participants were eliminated from the study.
2.4. Biochemical and Hematological Parameters
Blood samples were taken at the beginning and end of the intervention to determine selected biochemical and hematological parameters: blood lipids (total cholesterol, S-HDLC, serum low-density lipoprotein cholesterol (S-LDLC), triacylglycerols), hemocoagulation (aPTT, Quick test, INR, thrombocyte aggregation) and inflammatory parameters (TNF-α and IL-6). Analysis of all these parameters was performed using certified methods in the Department of Laboratory Diagnostics of Faculty Hospital Královské Vinohrady and Third Faculty of Medicine. Simultaneously, control measurements were performed of the blood pressure and heart rate.
2.5. Determining the Overall FA Profile in the Blood Plasma and in the Erythrocytes
Before analysis on a gas chromatograph (GC), fatty acid methyl ester (FAME) derivatives of all the samples were prepared. The FA profile in the plasma was determined using 200 µL of blood plasma, 100 µL of inert standard solution (IS) C13:0 (43 μg) and C17:0 (41 μg), and 4.2 mL of the derivatized mixture containing methanol/toluene/acetylchloride (3.2/0.8/0.2; v/v/v). Esterification was performed in a closed test tube for 1 h at 100 °C. Cooling was followed by neutralization with a 12% wt/v solution of K2CO3; the mixture was shaken for 10 min and centrifuged. The upper organic phase was then pipetted into a vial with an insert and 1 µL of sample was used for the analysis.
The FA composition of the erythrocyte membranes was determined by a slightly modified procedure according to Rose and Oklander [
35]. Approximately 8 mL of full blood taken with ethylenediaminetetraacetic acid (EDTA) was centrifuged at 1780 g for 5 min and, after removing the plasma, the obtained erythrocytes were rinsed with 3 mL of physiological solution. The test tube was then shaken for 2–3 min, centrifuged at 1780×
g for 4 min and the physiological solution was subsequently drawn off; this procedure was repeated four times. Subsequently, 1 mL of the rinsed erythrocytes was transferred to a closable test tube, 100 μL of methanol containing IS C13:0 (46 μg) and C17:0 (47 μg) were added and the solution was again shaken with 5 mL of isopropanol on a vortex shaker. The test tube was then shaken for 1 h; then 3.5 mL of chloroform was added and the mixture was again shaken for 1 h. This was followed by short centrifugation and the final extract was filtered and evaporated to dryness under a nitrogen stream at a temperature of 35 °C. The obtained lipids were stored in a freezer at −25 °C until esterification, which was performed by the same derivatization of the mixture as for the blood plasma.
All the FA determinations were performed on a GC-17A Version 3 gas chromatograph (Shimadzu, Kyoto, Japan) fitted with an AOC-20i autosampler modified for 15 samples with a standard flame ionization detector (FID). The temperatures of the injection port and FID were 250 °C and 260 °C, respectively. The basic FA profile was determined using a Stabilwax 15 m × 0.25 mm × 0.1 µm column (Restek, Bellefonte, PA, USA) and the carrier gas was helium with a flow rate of 1.2 mL/min. The temperature program began at 120 °C, followed by a gradient of 10 °C/min to 190 °C and a final gradient of 40 °C/min to 250 °C, which was maintained for 15.4 min. The splitting ratio for analysis of total FA and phospholipids from the erythrocytes was 1:60 and 1:30. The limit of quantification (LOQ) for the individual FA of the employed GC-FID technology varies in the range 1.7–8.3 μg/mL of blood plasma or erythrocytes.
The t-test was used for statistical processing of the set of data and the Excel program was employed to calculate parameter p. A value of p < 0.05 was taken as a statistically significant difference.
4. Conclusions
The addition of n-3 PUFA-rich flaxseed oil to the feed mixture demonstrably increases the content of especially ALA, and also of EPA and DHA in chicken and eggs, and is an effective technology for general production of n-3 FA foodstuffs. Regular eight-week consumption of omega-3 eggs and meat causes demonstrable changes in the erythrocyte phospholipid membranes, which are a long-term indicator of FA intake in foodstuffs. Amongst short-term indicators, such as the plasma FA level, an elevated ALA level caused by the ingestion of the enriched food was found. While eight-week intake of omega-3 foods was manifested in an increase in the levels of some n-3 PUFA in the blood and erythrocyte membranes, no effect was found on the basic body parameters, such as body weight, fat content, BMI, and also on the plasma cholesterol level, HDL, LDL, blood clotting, and inflammation markers.
Replacing standard soy with flaxseed oil as a source of lipids in feed mixture for fatty chickens and laying hens will ensure an enrichment of n-3 PUFAs from 250 mg to 900 mg/100 g of meat, and from 110 mg to 190 mg/100 g of eggs. With normal consumption of four servings of fortified meat and four whole eggs per week, this intervention increase the total LC-PUFA intake by 37 mg per day, which represents 7-15% of the recommended daily dose. This intervention has no demonstrable effect on the basic body parameters, such as body weight, fat content, BMI, and also on the plasma cholesterol level, HDL, LDL, blood clotting, inflammation markers, and omega-3 index.