Abstract
(FAs) are an essential component of the erythrocyte membrane, and nutrition and physical exercise are two variables that affect their structure and function. The aim of this study was to evaluate the erythrocyte profile in a group of high-level endurance runners, as well as the changes in different FAs, throughout a sports season in relation to the training performed. A total of 21 high-level male endurance runners (23±4 years; height: 1.76±0.05) were evaluated at four different times throughout a sports season. The athletes had at least 5 years of previous experience and participated in national and international competitions. The determination of the different FAs was carried out by gas chromatography. The runners exhibited low concentrations of docosahexaenoic acid (DHA) and omega-3 index (INDω-3), as well as high values of stearic acid (SA), palmitic acid (PA), and arachidonic acid (AA), compared to the values of reference throughout
5 years of previous experience and participated in national and international competitions. The determination of the different FAs was carried out by gas chromatography. The runners exhibited low concentrations of docosahexaenoic acid (DHA) and omega-3 index (INDω-3), as well as high values of stearic acid (SA), palmitic acid (PA), and arachidonic acid (AA), compared to the values of reference throughout the study. In conclusion, training modifies the erythrocyte FA profile in high-level endurance runners, reducing the concentrations of polyunsaturated fatty acids (PUFAs) such as DHA and AA and increasing the concentrations of saturated fatty acids (SFAs) such as SA and the PA. High-level endurance runners should pay special attention to the intake of PUFAsω-3 in their diet or consider supplementation during training periods to avoid deficiency. Keywords:endurance training; fatty acids; arachidonic acid; docosahexaenoic acid; omega-3 1. Introduction Exercise intensity, duration, distribution throughout a season, and recovery periods are essential variables in prescribing training for endurance runners [1]. Suitable nutritional intake is fundamental for recovery processes, adaptation, and optimizing performance in competition [2]. The roles of carbohydrates and proteins in endurance athletes have been extensively documented, continuing to be a subject of interest for performance optimization and recovery [3,4]. However, lipid intake receives less consideration by endurance athletes [5]. Fatty acids (FAs) play a crucial role in the athletes’ organisms, serving as energy stores and precursors to certain hormones, participating in the immune system, and being a structural component of cell membranes [6,7]. It is known that the composition of FAs ingested through the diet and their metabolism in the body correlates with the FAs that are part of different structures such as erythrocyte membranes, playing a crucial role in their function [8,9]. Special interest has been given to polyunsaturated fatty acids (PUFAs) in athletes: essential fatty acids that must be introduced through the diet [10]. PUFAs nutritionally important for human health are members of the so-called omega-3 (ω-3) and omega-6 (ω-6) Nutrients2024,16, 1895.
acids (PUFAs) in athletes: essential fatty acids that must be introduced through the diet [10]. PUFAs nutritionally important for human health are members of the so-called omega-3 (ω-3) and omega-6 (ω-6) Nutrients2024,16, 1895.
Nutrients2024,16, 1895 2 of 13 families. Within theω-6 family, arachidonic acid (AA) forms proinflammatory eicosanoids, especially prostaglandins, thromboxanes (potent vasoconstrictor), and leukotrienes that, in excessive amounts, contribute to thrombus formation, atheromas, and inflammatory disorders [11]. In relation to theω-3 PUFA family, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) stand out for their beneficial effects.ω-3 intake increases prostacyclin concentrations, which have vasodilatory and platelet aggregation inhibitory properties, balancing the activity of proinflammatory eicosanoids [12]. Another benefit observed in athletes is an increase in cell membrane fluidity, improving oxygen diffusion to tissues, as fluidity is determined by the presence of PUFAs located on both sides of lipid bilayers [13]. Peoples et al. [14] found a reduction in myocardial oxygen demand during exercise in a group of well-trained cyclists when theirω-3 concentrations increased. Besides,ω-3 participates in muscle protein synthesis, promoting recovery in athletes [15], and may enhance immune system function [16]. In erythrocytes, values ofω-3 below 8% may have negative effects on their integrity, associated with a higher risk of cardiovascular issues [17]. Deficits in this rate have been documented in various groups of athletes [18,19]. Regular training induces changes and adaptations in hematological and biochemical parameters that can affect erythrocyte cell membranes [20]. Exercise generates reactive oxygen species (ROS) and free radicals that have a disruptive effect on cells, requiring repair [21]. Erythrocyte membranes contain a high amount of PUFAs, and together with high amounts of oxygen and heme iron, making them susceptible to lipid peroxidation (LP) and potential muscle damage [22]. The peroxidation process involves the oxidation of a FA, converting it into a radical that can oxidize adjacent molecules [23]. The erythrocyte lifespan is around 120 days, making them a suitable matrix for measurement, reflecting their composition and long-term adaptations [24]. The type of exercise is another factor that modifies the FA profile in different tis- sues [25], as it requires greater mobilization and oxidation of FAs among tissues as an energy substrate [26]. Moderate-intensity exercise has been reported to increase prostacy- clins and decrease thromboxanes, favoring vasodilation and improving the body’s immune and anti-inflammatory response [27]. However, high-intensity training
[24]. The type of exercise is another factor that modifies the FA profile in different tis- sues [25], as it requires greater mobilization and oxidation of FAs among tissues as an energy substrate [26]. Moderate-intensity exercise has been reported to increase prostacy- clins and decrease thromboxanes, favoring vasodilation and improving the body’s immune and anti-inflammatory response [27]. However, high-intensity training increases the gener- ation of superoxide radicals in the lipid bilayers of muscle mitochondria, increasing the likelihood of erythrocyte damage [28]. Additionally, excessive free radical formation and microtrauma from high-intensity exercise can elevate the inflammatory state [29]. Several studies have reported altered erythrocyte FA profiles in different groups of athletes from different sports specialties compared to control subjects and reference ranges [16,18,19,30,31]. However, few longitudinal studies have been conducted in athletes to observe changes in various FAs over a sports season. In a recent study, Peña et al. [32] confirmed changes in different FAs in a women’s soccer team over multiple seasons, a sport characterized by high levels of competitive activity and high-intensity training. Nevertheless, the periodization of loads and competitions in endurance runners differs significantly during the season. They have lower competitive density and train high volumes, with the majority at moderate–low intensity and a small percentage at high intensity, above the second ventilatory threshold (VT2) (1). We hypothesize that regular training will induce changes in erythrocyte FA percentages in runners, particularly during periods of increased competitive activity and high-intensity training. Therefore, this study aimed to determine the FA profile in erythrocytes in a group of high-level endurance runners at different points throughout a sports season and observe changes resulting from modifications in weekly training volumes and intensities during different periods. 2. Materials and Methods 2.1. Participants The study was carried out on 21 high-level male endurance runners (23±3 years old; height: 1.76±0.04 m; 65.50±7.30 kg) who resided within the same geographical region. They had personal bests between 3:37.79 to 4:08.24 in the 1500 m and from 13:11.01 to 15:10.35 in the 5000 m. Also, runners exhibited values over 65 mL/kg/min of VO2
was carried out on 21 high-level male endurance runners (23±3 years old; height: 1.76±0.04 m; 65.50±7.30 kg) who resided within the same geographical region. They had personal bests between 3:37.79 to 4:08.24 in the 1500 m and from 13:11.01 to 15:10.35 in the 5000 m. Also, runners exhibited values over 65 mL/kg/min of VO2
Nutrients2024,16, 1895 3 of 13 max [33]. The study sample was the same as in previous research [34,35]. Four samplings were carried out every 3 months during a sports season. All the subjects were healthy and had to meet the following inclusion criteria: (i) to be men; (ii) to compete at the national and international level in endurance races; (iii) to have at least 5 years of competing experience; (iv) to carry out more than 5 weekly training sessions and 70 km per week during the season; (v) not to change nutritional habits during the research; (vi) no weight changes >3% during the season. The exclusion criteria were: (i) not completing 25% of the training sessions due to injury or illness and (ii) ingestingω-3 supplements during the research or any supplements in the three weeks previous to the samples’ collection. Informed written consent was secured from all participants. The study protocol re- ceived approval from the Ethics Committee of the University of Extremadura (52/2012) and was conducted in accordance with the principles outlined in the 1975 Helsinki Declara- tion, as revised by the World Medical Assembly in Fortaleza, Brazil, in 2013, for research involving human subjects. 2.2. Study Design Four samples were taken from the runners during a sports season, one every 3 months. The initial evaluation (Initial) was carried out in the first week of October, after an adapta- tion period of about 2–3 weeks when the runners only performed sessions below VT2, since they had had a rest period of between 2–3 weeks without training at the end of the previous season. In the first week of January, the second evaluation (3 months) was performed after completing the first preparatory period from October to December. The third evaluation (6 months) was carried out the first week of April, after finishing the first competitive period when the athletes carried out cross-country competitions. Finally, in the first week of July, the fourth evaluation (9 months) was performed, when the runners had completed a second but smaller preparatory period between April and May and were in the second competitive period that
(6 months) was carried out the first week of April, after finishing the first competitive period when the athletes carried out cross-country competitions. Finally, in the first week of July, the fourth evaluation (9 months) was performed, when the runners had completed a second but smaller preparatory period between April and May and were in the second competitive period that took place between June and July when the runners competed in a track-field competition. After the first competitive period, the runners had 2 weeks of transition, with less volume and without high intensity sessions. 2.3. Nutritional Assessment The nutritional evaluation was carried out following the same methodology used in previous studies by our research group [36]. All participants completed a 3-day dietary record using the provided nutritional questionnaire, which included one weekend day and two weekdays. Each participant meticulously weighed and recorded the amount of each food item consumed in grams. The athletes’ dietary intake was analyzed using a comprehensive food composition table [37]. Table during the season. The athletes adhered to a diet formulated according to established energy and macronutrient guidelines [38]. Table 1.Nutritional intake of lipids and fatty acids during the season. Parameters INITIAL 3 MONTHS 6 MONTHS 9 MONTHS Lipids (g/kg/d) 1.82 ±0.85 1.42 ±0.57 1.40 ±0.44 1.77 ±0.86 Saturated fatty acids (g/day) 37.45 ±21.14 31.45 ±11.14 33.45 ±14.54 36.15 ±19.17 Monounsaturated fatty acids (g/day) 52.27±25.24 40.76 ±17.24 38.03 ±12.24 43.45 ±22.18 Polyunsaturated fatty acids (g/day) 11.97±5.35 12.11 ±4.69 11.56 ±3.93 13.08 ±8.58 ω-6 (g/day) 9.28 ±4.82 9.08 ±4.84 9.47 ±4.00 10.58 ±6.34 ω-3 (g/day) 1.08 ±0.44 0.98 ±0.53 1.03 ±0.44 1.33 ±1.11 2.4. Anthropometric and Ergoespirometric Measures Table the runners during the season. Anthropometric measurements were performed on athletes
Nutrients2024,16, 1895 4 of 13 with an empty stomach and always at the same time, between 9 and 10 a.m. Body weight (Seca 769, Hamburg, Germany) and height (Seca 220, Hamburg, Germany) were measured to the nearest 0.1 kg and 0.1 cm, respectively, in accordance with the manufacturer’s guidelines. Six skinfolds (subscapular, triceps, supraspinal, abdominal, thigh, and calf) were assessed using a skinfold caliper (Holtain, Crosswell, UK) with a precision of 0.1 mm. Each skinfold measurement was taken three times, with the average value recorded in millimeters. All measurements were conducted by an experienced and certified tester following the International Society for the Advancement of Kinanthropometry (ISAK) protocol. Fat and muscle weight were calculated following the instructions of ISAK [39]. Table 2.Anthropometric and ergoespirometrics characteristics in the runners during the season. Parameters INITIAL 3 MONTHS 6 MONTHS 9 MONTHS VO 2max (mL/kg/min) 68.30 ±4.45 67.82 ±8.23 68.80 ±6.73 68.62 ±7.37 VT 2(%VO 2max) 90.84 ±2.68 92.56 ±3.27 91.04 ±3.44 90.71 ±2.05 vVT 2(Km/h) 19.37 ±0.90 20.08 ±0.80 ** 19.76 ±1.10 19.48 ±1.40 Maximum heart rate (bpm) 190 ±9 192 ±7 194 ±9 193 ±7 Weight (kg) 65.50 ±7.30 65.45 ±7.36 64.67 ±7.03 * 64.80 ±7.34 * Fat mass (kg) 5.59 ±1.23 5.42 ±1.07 5.24 ±0.83 * 5.24 ±0.96 * Muscle mass (kg) 32.19 ±4.00 32.36 ±4.01 31.83 ±3.93 31.88 ±4.12 * VO2max: maximal oxygen consumption; VT2: second ventilatory threshold; vVT2: running speed at second ventilatory threshold; bpm: beats per minute. *p< 0.05 initial vs. 3, 6, 9 months. **p< 0.05 initial vs. 3, 6, 9 months. After recording anthropometric measurements, the runners carried out an incremental treadmill test to exhaustion (Powerjog, Birmingham, UK) to assess their ergospirometric parameters and performance. This test was conducted using an ergospirometer system with a gas analyzer (Metamax, Cortex Biophysik, Leipzig, Germany). Additionally, a heart rate monitor (Vantage M, Polar, Finland) was employed to measure the maximal heart rate. After a 10 min warm-up, the runners began the test at a speed of 10 km/h, with the speed increasing by 1 km/h every 400 m until they reached voluntary exhaustion. VO2 max was determined based
gas analyzer (Metamax, Cortex Biophysik, Leipzig, Germany). Additionally, a heart rate monitor (Vantage M, Polar, Finland) was employed to measure the maximal heart rate. After a 10 min warm-up, the runners began the test at a speed of 10 km/h, with the speed increasing by 1 km/h every 400 m until they reached voluntary exhaustion. VO2 max was determined based on the following criteria: a plateau in oxygen uptake (VO2), an increase in carbon dioxide (CO2) elimination, an increase in ventilatory volume (VE) due to the increased test velocity, and a respiratory exchange ratio (RER) exceeding 1 [40]. The aerobic threshold VT1and VT2were identified according to the three-phase model for monitoring training and internal load [41]. 2.5. Training Characteristics A GPS package equipped with a heart rate monitor (Polar Electro, Polar Vantage M. Kempele, Finland) was used to track training loads throughout the season. Runners quantified the internal load through total time in each training zone [1]. Table the training characteristics of the runners. The athletes engaged in two to three resistance training sessions per week throughout the athletics season. Generally, the exercise volume was moderate to high (2–4 sets of 4–16 repetitions) while the intensity was low–moderate (30–70% of 1RM). Table 3.Training characteristics in the runners during the season.INITIAL 3 MONTHS 6 MONTHS 9 MONTHS Training (km/week) 44.32 ±8.16 114.78 ±18.26 101.11 ±15.54 80.90 ±13.36 <VT 2(km/week) 44.32 ±8.16 91.83 ±14.61 75.83 ±11.66 69.62 ±11.36 >VT 2(km/week) - 22.96 ±3.65 25.28 ±3.89 12.29 ±2.01 VT2: anaerobic threshold. 2.6. Sample Collection Following the anthropometric assessment of the athletes, venous blood samples were drawn from the antecubital vein into 10 mL tubes containing Ethylene Diamine Tetra
Nutrients2024,16, 1895 5 of 13 Acetic Acid (EDTA). These samples were immediately centrifuged at 3000 rpm for 10 min. Subsequently, erythrocytes were washed thrice with 0.9% sodium chloride (NaCl). The erythrocytes were then transferred to sterile tubes and stored at−80 ◦ C until analysis. 2.7. Analytical Determination FA concentrations were determined in erythrocytes using the technique described by Lepage and Roy [42]. A gas chromatograph HP-5890 Series II equipped with a Flame Ionisation Detector (FID) was utilized. The analysis was conducted on a BP×70 capillary column (50 m×0.22 mm I.D., 0.25µm film thickness, Cromlab, Barcelona, Spain). The initial oven temperature was set to 170 ◦ C and maintained for 15 min. It was then increased to 190 ◦ C at a rate of 3 ◦ C/min and held for 15 min, followed by an increase to 245 ◦ C at 3 ◦ C/min, with the final temperature held for 30 min. Helium (He) was used as the carrier gas at a flow rate of 1.0 mL/min. The injector operated in splitless mode at 300 ◦ C, with a purge flow of 6 mL/min applied 0.5 min post-injection. The FID was maintained at 250 ◦ C. FA identification was achieved by comparing the retention times of the FA methyl esters with those of known FA standards under identical chromatographic conditions, using retention parameters relative to an internal standard. Heptadecanoic acid was selected as the internal standard due to its similarity to the analyses and its distinct chromatographic position, which did not overlap with other sample peaks. FA concentrations were expressed as a percentage of total FAs (relative %), with over 97% of GC peaks being accurately identified using appropriate standards. 2.8. Lipid Profile of the Erythrocyte Membranes Ten FAs were selected for analysis: for Saturated Fatty Acids (SFAs), palmitic acid (PA) and stearic acid (SA); for Monounsaturated Fatty Acids (MUFAs), oleic acid (OA); forω-3 PUFAs, alpha-linolenic acid (ALA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA); forω-6 PUFAs, linoleic acid (LA), calendic acid (CA), and AA. Based on these FAs, various indices were calculated: the Saturation Index (SI), calculated
for analysis: for Saturated Fatty Acids (SFAs), palmitic acid (PA) and stearic acid (SA); for Monounsaturated Fatty Acids (MUFAs), oleic acid (OA); forω-3 PUFAs, alpha-linolenic acid (ALA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA); forω-6 PUFAs, linoleic acid (LA), calendic acid (CA), and AA. Based on these FAs, various indices were calculated: the Saturation Index (SI), calculated as the ratio of % SFAs to % MUFAs, relating to membrane fluidity; theω-3 index (ω-3 IND) as the sum of DHA and EPA; the inflammatory risk index as the ratio of %ω-6 to %ω-3. Additionally, enzymatic indices for elongase (SA/PA),ω-3 desaturase (DHA/DPA), and delta-9 desaturase (OA/SA) activities were calculated. Optimal value ranges for each of the ten FAs were derived from existing literature [24]. 2.9. Statistical Analysis The statistical analysis was conducted using IBM SPSS Statistics software version 21.0 (IBM Co., Armonk, NY, USA). The results are presented as x±s, where x represents the mean values and s denotes the standard deviation. Prior to analysis, all variables were tested for normality using Kolmogorov–Smirnov tests. Data were analyzed via repeated measures analysis of variance (ANOVA) with the Bonferroni post hoc test for mo- ment/period as the categorical variable. The equality of variances between differences was evaluated using Mauchly’s test of sphericity. When sphericity was violated, Greenhouse– Geisser correctedp-values were applied. Simple linear regression analysis was performed to investigate associations between FAs, theω-3 index, theω-6/ω-3 ratio, and kilometers trained per week. Ap-value of less than 0.05 was considered statistically significant. 3. Results The profile of fatty acids in erythrocytes in the runners throughout the sports season is shown in Table. Within the SFAs, we can observe a very significant increase ( p< 0.01) in PA at 3 and 9 months and significant (p< 0.05) at 6 months compared to the beginning of the season. A significant decrease also occurred between 3 and 6 months. In relation to SA, we found an increase (p< 0.05) at 3 months compared to the beginning and a decrease at 6 months compared to 3 months.
Description
The research examines how training affects fatty acid profiles in elite runners.