Increased Plasma L-Arginine Levels and L-Arginine/ADMA Ratios after Twelve Weeks of Omega-3 Fatty Acid Supplementation in Amateur Male Endurance Runners

is not fully understood how supplementation with omega-3 fatty acids affects the metabolism of amino acids required for the bioavailability/synthesis of NO, i.e., L-arginine (L-arg), asymmetric dimethylarginine (ADMA), their metabolites, and the L-arg/ADMA ratio and their impact on running economy (RE) in runners. Thus, 26 male amateur endurance runners completed a twelve-week study in which they were divided into two supplemented groups: the OMEGA group (n= 14; 2234 mg and 916 mg of eicosapentaenoic and docosahexaenoic acid daily) or the MCT group (n= 12; 4000 mg of medium-chain triglycerides daily). At the same time, all participants followed an endurance training program. Before and after the 12-week

amateur endurance runners completed a twelve-week study in which they were divided into two supplemented groups: the OMEGA group (n= 14; 2234 mg and 916 mg of eicosapentaenoic and docosahexaenoic acid daily) or the MCT group (n= 12; 4000 mg of medium-chain triglycerides daily). At the same time, all participants followed an endurance training program. Before and after the 12-week intervention, blood was collected from participants at two time points (at rest and immediately post-exercise) to determine EPA and DHA in red blood cells (RBCs) and plasma levels of L-arg, ADMA, and their metabolites. RBC EPA and DHA signi cantly increased in the OMEGA group (p< 0.001), which was related to the resting increase in L-arg (p= 0.001) and in the L-arg/ADMA ratio (p= 0.005) with no changes in the MCT group. No differences were found in post-exercise amino acid levels. A total of 12 weeks of omega-3 fatty acid supplementation at a dose of 2234 mg of EPA and 916 mg of DHA daily increased levels of L-arg and the L-arg/ADMA ratio, which indirectly indicates increased bioavailability/NO synthesis. However, these changes were not associated with improved RE in male amateur endurance runners. Keywords: omega-3 fatty acids; L-arginine; ADMA; nitric oxide; running economy; endurance runners 1. Introduction Supplementation with omega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has effects that include, but are not limited to, a reduction in the risk of cardiovascular diseases [1,2], nervous system diseases [3] and metabolic diseases such as diabetes mellitus [4]. Moreover, in healthy, trained and/or untrained subjects, supplementation with omega-3 fatty acids has been shown to enhance muscle function and recovery [5,6]. Evidence for performance improvement in endurance athletes following omega-3 fatty acid supplementation is scarce; however, our recent study showed that 12-week supplementation with omega-3 fatty acids in amateur runners increased the so-called omega-3 index (O3I) (expressed as a sum of % EPA and % DHA levels in red blood cells (RBCs)) which was associated with improved running economy (RE) [7]. Nonetheless, the underlying mechanism appears to be complex and is not fully understood. Among the proposed mechanisms is

showed that 12-week supplementation with omega-3 fatty acids in amateur runners increased the so-called omega-3 index (O3I) (expressed as a sum of % EPA and % DHA levels in red blood cells (RBCs)) which was associated with improved running economy (RE) [7]. Nonetheless, the underlying mechanism appears to be complex and is not fully understood. Among the proposed mechanisms is an increase in the release of nitric oxide (NO) by the vascular Nutrients2022,14, 4749.

Nutrients2022,14, 4749 2 of 10 endothelium, which is characteristic of, among others, aerobic physical training [8]. This phenomenon is possibly due to the metabolism of L-arginine (L-arg) into L-citrulline via endothelial nitric oxide synthase (eNOS); among the products of this transformation is NO [9]. As a result, there is an increase in cyclic guanosine monophosphate (cGMP), which leads to the relaxation of smooth muscle and vasodilation [10]. On the other hand, the vasodilator effect is antagonized in the presence of asymmetric dimethylarginine (ADMA) in plasma, a competitive inhibitor for eNOS [11,12]. Both ADMA and the second amino acid from the methylarginase family, symmetric dimethylarginine (SDMA) negatively correlate with the bioavailability of NO, although the latter weakly and indirectly inhibits NO synthesis [13]. Increased plasma ADMA and/or SDMA levels are related to an impairment of vascular functions, thus becoming a factor increasing the risk of cardiovascular diseases [14,15]. Previous research suggests the L-arg/ADMA ratio as among the robust tools for assessing vascular endothelial function [16]. Low values of the ratio increase the risk of impaired vascular endothelial function, and therefore enhance the rate of hospitalization and mortality [17]. Decreased levels of L-arg and a lower L-arg/ADMA ratio observed after strenuous exercise may result in reduced ability to synthesize NO [18]. Hence, nding an exogenous modulator of these amino acids seems to be important not only for the sedentary, but also for healthy, physically active people and athletes. Despite the positive effect of supplementation with omega-3 fatty acids on the exercise capacity of endurance athletes [19,20], de ciencies of omega-3 fatty acids are still observed, among others, in the diet of NCAA athletes [21]. Mechanisms responsible for changes in amino acid metabolism following supplemen- tation with omega-3 fatty acids are not comprehensively understood, and the effect on L-arg metabolites and the L-arg/ADMA ratio seems to be crucial in understanding the effect of omega-3 fatty acids among athletes. Thus, the aim of this study was twofold— rstly, to investigate the effect of 12-week supplementation with omega-3 fatty acids on the plasma levels of L-arg, ADMA, the L-arg/ADMA ratio and related metabolites and, secondly,

understood, and the effect on L-arg metabolites and the L-arg/ADMA ratio seems to be crucial in understanding the effect of omega-3 fatty acids among athletes. Thus, the aim of this study was twofold— rstly, to investigate the effect of 12-week supplementation with omega-3 fatty acids on the plasma levels of L-arg, ADMA, the L-arg/ADMA ratio and related metabolites and, secondly, to assess whether the aforementioned markers correlate with RE in male amateur endurance athletes. 2. Materials and Methods 2.1. Participants Twenty-six male runners (37 3 years old; 77 9 kg body weight; VO 2peak: 54.2 6 mL*kg 1 *min 1 ) completed a randomized controlled trial, approved by the Bioeth- ical Committee of Regional Medical Society in Gda ´nsk (NKBBN/628/2019) and conducted according to the Declaration of Helsinki. 2.2. Study Design This study was part of a larger research project with details outlined elsewhere [7], and characteristics of the participants are shown in Table. Brie y, participants were randomly assigned to one of two groups with the nal characteristics as follows: OMEGA (age: 37 3 years; body weight: 76 11 kg; VO 2peak: 53.8 5 mL*kg 1 *min 1 ) or medium-chain triglycerides (MCT) (age: 37 4 years; body weight: 78 8 kg; VO 2peak: 54.7 7 mL*kg 1 *min 1 ). All participants completed a 12-week programme that in- cluded 4 training sessions per week (3 running sessions + 1 core strengthening ses- sion). The training structure was based on the ventilatory threshold (VT) and ventilatory anaerobic threshold (VAT) method with corresponding three heart rate (HR) zones: [Z1: HR@VT1 + 5 bpm;Z2: (>HR@VT1 + 5 bpm) to ( HR@VAT-5 bpm); Z3: >HR@VAT-5 bpm]. Simultaneously, participants ingested 4 capsules per day providing a total of2234 mg of EPA + 916 mg of DHA (OMEGA group) or 4000 mg of MCTs (MCT group). Before and after the 12-week period, VO 2peakduring an incremental treadmill test was measured on a motorized treadmill (h/p Cosmos, Saturn, Germany) and blood samples were taken twice: before starting and immediately after nishing the test. The test consisted of a few stages: rst, participants walked for 5

mg of DHA (OMEGA group) or 4000 mg of MCTs (MCT group). Before and after the 12-week period, VO 2peakduring an incremental treadmill test was measured on a motorized treadmill (h/p Cosmos, Saturn, Germany) and blood samples were taken twice: before starting and immediately after nishing the test. The test consisted of a few stages: rst, participants walked for 5 min at 5 km/h speed and with a 1.5% incline as a warm-up.

Nutrients2022,14, 4749 3 of 10 Second, the treadmill belt was accelerated starting from 8 km/h by 1 km/h per stage up to 12 km/h with every next stage duration of 3 min. Then, the incline of the treadmill was increased to 5%, 10% and 15% at 12 km/h speed until volitional exhaustion. During both tests, heart rate (HR) was monitored (Polar RS400, Kempele, Finland). Additionally, oxygen uptake (VO2), carbon dioxide output (VCO2), minute ventilation (Ve) and respira- tory exchange ratio (RER) were continuously measured using a breath-by-breath analyzer (Oxycon Pro, Jaeger, Hoechberg, Germany). VO 2peakwas obtained as the highest 30 s mean value recorded during the test. RE was measured as an oxygen cost from last 50 s as previously described [22] with slight modi cations accordingly toTomczyk et al., 2022 [7]. Table 1.Characteristics of participants. Variable MCT (n= 12) Mean SD OMEGA (n= 14) Mean SD Age (years) 37 4 37 3 Body mass (kg) 78 8 76 11 Height (cm) 180 4 181 7 VO 2peak(mL*kg 1 *min 1 ) 54.7 7 53.6 4 RE (mL*kg 1 *min 1 ) Pre 47.7 3.3 Pre 47.6 1.8 Post 48.7 2.9 Post 46.5 2.4 † EPA (% of total RBC fatty acids) Pre 1.2 0.3 Pre 1.1 0.4 Post 1.2 0.3 Post 4.9 1.1 * † DHA (% of total RBC fatty acids) Pre 4.4 1.1 Pre 4.7 1.0 Post 4.5 0.8 Post 6.7 0.8 * † O3I Pre 5.6 1.4 Pre 5.8 1.3 Post 5.6 1.1 Post 11.6 1.7 * † Test duration (min: s) Pre 1091 144 Pre 1111 70 Post 1137 84 * Post 1138 85 *p< 0.05 post vs. pre; † p< 0.05 MCT vs. OMEGA; SD—standard deviation; EPA—eicosapentaenoic acid; DHA—docosahexaenoic acid; RBC—red blood cell; O3I—Omega-3 index. 2.3. Sample Collection Blood samples were collected into 4 mL sodium citrate vacutainer tubes and cen- trifuged at 4 C (4000 gfor 10 min). After centrifugation, plasma and RBCs were collected with a disposable Pasteur pipette and transferred into separate Eppendorf probes and stored in a 80 C freezer until further analysis. 2.4. Fatty Acid Analysis Concentrations of EPA

index. 2.3. Sample Collection Blood samples were collected into 4 mL sodium citrate vacutainer tubes and cen- trifuged at 4 C (4000 gfor 10 min). After centrifugation, plasma and RBCs were collected with a disposable Pasteur pipette and transferred into separate Eppendorf probes and stored in a 80 C freezer until further analysis. 2.4. Fatty Acid Analysis Concentrations of EPA and DHA in red blood cells (RBCs) were measured using gas chromatography [23]. Brie y, RBC lipids were extracted into chloroform methanol and fatty acid methyl esters (representing the RBC fatty acids) were formed by heating the lipid extract with methanolic sulphuric acid. The fatty acid methyl esters were separated by gas chromatography on a Hewlett Packard 6890 gas chromatograph tted with a BPX-70 column. Fatty acid methyl esters were identi ed by comparison with run times of authentic standards. Fatty acids are expressed as a % of total fatty acids present. 2.5. Amino Acid Assessment Determinations of plasma L-arginine, ornithine, L-citrulline, DMA, ADMA and SDMA concentrations were performed using high-performance liquid chromatography with tan- dem mass spectrometry (LC-MS/MS) with prior protein precipitation and derivatization. To 50 L of plasma, 200 L of protein precipitation reagent was added (mixture of internal standards in water and methanol, 20:80). The sample was stirred for 15 min (1100 grpm ) and centrifuged (3000 grpm, 10 min). A volume of 10 L of supernatant was trans- ferred to a new insert vial and subjected to AccQ-Tag (Waters Co, Milford, MA, USA) derivatization in accordance with the manufacturer's recommendations. After derivatiza-

Nutrients2022,14, 4749 4 of 10 tion, samples were diluted 1:1 with ultrapure water and subjected to LC-MS/MS analysis accordingly to Carling et al. [24] with slight modi cations. 2.6. Statistical Analysis Statistical analysis was performed using GraphPad Prism 7. Each variable was sub- jected to normal distribution analysis using the Shapiro–Wilk test. Arithmetic means, standard deviation and signi cance levels were calculated. When the distribution of the variable was normal, the pairedt-test was used, while when the distribution was not normal the non-parametric Wilcoxon test was used. Then, two-way analysis of variance (ANOVA) with repeated measures to investigate the signi cance of differences between groups and time was used. Signi cant main effects were further analyzed using the Sidak post hoc test. Correlations between variables were evaluated using the Spearman correlation coef cient. Signi cance for all analyses was assumed atp< 0.05. 3. Results 3.1. Omega-3 Polyunsaturated Fatty Acids in RBCs Baseline levels of EPA and DHA and the O3I did not differ between the two groups (OMEGA group: 1.1% EPA, 4.7% DHA, 5.8% O3I; MCT group: 1.2% EPA, 4.4% DHA, 5.6% O3I, allp> 0.999). Post-intervention values of EPA, DHA and O3I increased in the OMEGA group to 4.9% EPA, 6.7% DHA, 11.6% O3I (allp< 0.001). Changes were not observed in the MCT group (1.2% EPA,p> 0.999; 4.7% DHA,p= 0.551; 5.8% O3I,p> 0.999). 3.2. Plasma L-arginine and Its Metabolites at Resting Conditions The plasma levels of L-arg and its metabolites for both groups at rest are provided in Table. For L-arg, a statistically signi cant increase was noted in the OMEGA group (p= 0.001), while there was no change (p= 0.109) in the MCT group after 12 weeks of supplementation. The level of ornithine was signi cantly decreased from pre to post in both groups (p< 0.001 andp= 0.007 for the OMEGA and MCT groups, respectively). Additionally, the L-arg/ADMA ratio was increased in the OMEGA group from pre to post (p= 0.005), while there was no change in the MCT group (p= 0.077).Nutrients 2022, 14, x FOR PEER REVIEW 4 of 11 2.5. Amino Acid Assessment Determinations of plasma L-arginine,

to post in both groups (p< 0.001 andp= 0.007 for the OMEGA and MCT groups, respectively). Additionally, the L-arg/ADMA ratio was increased in the OMEGA group from pre to post (p= 0.005), while there was no change in the MCT group (p= 0.077).Nutrients 2022, 14, x FOR PEER REVIEW 4 of 11 2.5. Amino Acid Assessment Determinations of plasma L-arginine, ornithine, L-citrulline, DMA, ADMA and SDMA concentrations were performed using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) with prior protein precipitation and deri- vatization. To 50 µL of plasma, 200 µL of protein precipitation reagent was added (mix- ture of internal standards in water and methanol, 20:80). The sample was stirred for 15 min (1100× g rpm) and centrifuged (3000× g rpm, 10 min). A volume of 10 µL of superna- tant was transferred to a new insert vial and subjected to AccQ-Tag (Waters Co, Milford, MA, USA) derivatization in accordance with the manufacturer’s recommendations. After derivatization, samples were diluted 1:1 with ultrapure water and subjected to LC-MS/MS analysis accordingly to Carling et al. [24] with slight modifications. 2.6. Statistical Analysis Statistical analysis was performed using GraphPad Prism 7. Each variable was sub- jected to normal distribution analysis using the Shapiro–Wilk test. Arithmetic means, standard deviation and significance levels were calculated. When the distribution of the variable was normal, the paired t-test was used, while when the distribution was not nor- mal the non-parametric Wilcoxon test was used. Then, two-way analysis of variance (ANOVA) with repeated measures to investigate the significance of differences between groups and time was used. Significant main effects were further analyzed using the Sidak post hoc test. Correlations between variables were evaluated using the Spearman correla- tion coefficient. Significance for all analyses was assumed at p < 0.05. 3. Results 3.1. Omega-3 Polyunsaturated Fatty Acids in RBCs Baseline levels of EPA and DHA and the O3I did not differ between the two groups (OMEGA group: 1.1% EPA, 4.7% DHA, 5.8% O3I; MCT group: 1.2% EPA, 4.4% DHA, 5.6% O3I, all p > 0.999). Post-intervention values of EPA, DHA and O3I increased in the OMEGA group

p < 0.05. 3. Results 3.1. Omega-3 Polyunsaturated Fatty Acids in RBCs Baseline levels of EPA and DHA and the O3I did not differ between the two groups (OMEGA group: 1.1% EPA, 4.7% DHA, 5.8% O3I; MCT group: 1.2% EPA, 4.4% DHA, 5.6% O3I, all p > 0.999). Post-intervention values of EPA, DHA and O3I increased in the OMEGA group to 4.9% EPA, 6.7% DHA, 11.6% O3I (all p < 0.001). Changes were not ob- served in the MCT group (1.2% EPA, p > 0.999; 4.7% DHA, p = 0.551; 5.8% O3I, p > 0.999). 3.2. Plasma L-arginine and Its Metabolites at Resting Conditions The plasma levels of L-arg and its metabolites for both groups at rest are provided in Table 2 and Figure 1. For L-arg, a statistically significant increase was noted in the OMEGA group (p = 0.001), while there was no change (p = 0.109) in the MCT group after 12 weeks of supplementation. The level of ornithine was significantly decreased from pre to post in both groups (p < 0.001 and p = 0.007 for the OMEGA and MCT groups, respec- tively). Additionally, the L-arg/ADMA ratio was increased in the OMEGA group from pre to post (p = 0.005), while there was no change in the MCT group (p = 0.077). Figure 1. Resting plasma L-arginine (A) and ornithine (B) levels and L-arginine/ADMA ratios (C) pre- and post-12 weeks of supplementation (* p < 0.05- pre vs. post). Figure 1. Resting plasma L-arginine (A) and ornithine (B) levels and L-arginine/ADMA ratios (C) pre- and post-12 weeks of supplementation (*p< 0.05- pre vs. post). Table 2. The effect of 12-week omega-3 fatty acid supplementation on resting plasma levels of L-arginine and its metabolites. MCT (n= 12) Mean SD OMEGA (n= 14) Mean SD Diff 95% CI p Lower Upper L-arginine ( mol/L) Before 109.4 17.53 105.4 14.67 4.003 17.4 9.394 0.744 After 120.4 15.55 122.0 11.12 1.621 11.78 15.02 0.952 Change 11.00 17.21 16.63 14.87 p 0.109 0.001

SD OMEGA (n= 14) Mean SD Diff 95% CI p Lower Upper L-arginine ( mol/L) Before 109.4 17.53 105.4 14.67 4.003 17.4 9.394 0.744 After 120.4 15.55 122.0 11.12 1.621 11.78 15.02 0.952 Change 11.00 17.21 16.63 14.87 p 0.109 0.001

The study investigates the effects of omega-3 supplementation on plasma L-arginine levels in runners.