Krill-Oil-Dependent Increases in HS-Omega-3 Index, Plasma Choline and Antioxidant Capacity in Well-Conditioned Power Training Athletes

idence that both omega-3 polyunsaturated fatty acids (n-3 PUFAs) and choline can in uence sports performance, but information establishing their combined effects when given in the form of krill oil during power training protocols is missing. The purpose of this study was therefore to characterize n-3 PUFA and choline pro les after a one-hour period of high-intensity physical workout after 12 weeks of supplementation. Thirty- ve healthy power training athletes received either 2.5 g/day of Neptune krill oil TM (550 mg EPA/DHA and 150 mg choline) or olive oil (placebo) in a randomized double-blind design. After 12 weeks, only

this study was therefore to characterize n-3 PUFA and choline pro les after a one-hour period of high-intensity physical workout after 12 weeks of supplementation. Thirty- ve healthy power training athletes received either 2.5 g/day of Neptune krill oil TM (550 mg EPA/DHA and 150 mg choline) or olive oil (placebo) in a randomized double-blind design. After 12 weeks, only the krill oil group showed a signi cant HS-Omega-3 Index increase from 4.82 to 6.77% and a reduction in the ARA/EPA ratio (from 50.72 to 13.61%) (p< 0.001). The krill oil group showed signi cantly higher recovery of choline concentrations relative to the placebo group from the end of the rst to the beginning of the second exercise test (p= 0.04) and an 8% decrease in total antioxidant capacity post-exercise versus 21% in the placebo group (p= 0.35). In conclusion, krill oil can be used as a nutritional strategy for increasing the HS-Omega-3 Index, recover choline concentrations and address oxidative stress after intense power trainings. Keywords: choline; CrossFit TM ; power training; DHA; EPA; high-intensity interval training; krill oil; HS-Omega-3 Index; oxidative stress; phosphatidylcholine; sports nutrition 1. Introduction The high-intensity modality of exercise performed during short periods of time has gained popularity in the last decades due to factors related to health, fashion and a sense of well-being. These activities are known as power training (PT) sports, or high-intensity interval training activities (HIIT), but sometimes popularly referred to as CrossFit, which is a registered trademark (CrossFit TM ). Whereas power lifting focuses on bench press, squat and deadlift, the PT schedule is characterized by functional movements performed at high intensity, combining intervals of strength and endurance that can vary between weightlifting, running, cycling, squatting, pulling, pushing, etc. with little or no rest in between [1,2]. Because this functional movement training method is metabolically very demanding, its bene cial effect on health and on improving athletic performance has been contradictorily assessed [3–6]. Since risks for prolonged responses of oxidative Nutrients2021,13, 4237.

no rest in between [1,2]. Because this functional movement training method is metabolically very demanding, its bene cial effect on health and on improving athletic performance has been contradictorily assessed [3–6]. Since risks for prolonged responses of oxidative Nutrients2021,13, 4237.

Nutrients2021,13, 4237 2 of 16 stress and in ammation as well as injury and overreaching have been highlighted, dietary interventions may be required to improve athletic performance and speed up recovery. There are some studies available that evaluated the bene ts of nutritional interventions in PT athletes [7–13]. However, no interventional data exist that establish choline and omega-3 polyunsaturated fatty acid (n-3 PUFA) intake as a supplementation strategy to optimize PT training sessions and recovery, and these important nutrients seem so far to go unnoticed in this sporting discipline [14]. Given that 90% of the US population is known to consume too little choline [15], and that European populations also do not reach the recommended daily intake levels of 400 mg [16], this indicates that athletes might also be at risk of choline de ciency. The body can produce choline in limited amounts by using one-carbon groups from the folate metabolism, but most of it has to be supplied by the diet [17]. It is noteworthy that a person's need for endogenous choline is increased if genetic modi cations of genes involved in the folate metabolism are present, which will further increase the risk for choline de ciency [18]. During exercise, plasma choline is needed as a precursor for acetylcholine, which is a neurotransmitter responsible for muscles contractions [19–21]. If not enough plasma choline is available, then the body can resort to breaking down phosphatidylcholine, the building blocks of cell membranes, which can compromise membrane integrity and stress resistance [22,23]. Vulnerable membranes and reduced acetylcholine production during intense physical exercise might promote muscle damage and decrease muscle stimulation resulting in muscle exhaustion [17]. In addition to being essential for neurotransmitter synthesis, choline is involved in cell-membrane signaling, transport of fat and methyl group metabolism, which are all important functions for optimal sports performance [24]. However, plasma choline concentrations are known to be challenged during strenuous physical exercise. In particular, in endurance athletes performing more than two hours of intense, physical activity at more than 70% VO2max, a signi cant decrease of plasma choline concentrations has been described [17]. In both

and methyl group metabolism, which are all important functions for optimal sports performance [24]. However, plasma choline concentrations are known to be challenged during strenuous physical exercise. In particular, in endurance athletes performing more than two hours of intense, physical activity at more than 70% VO2max, a signi cant decrease of plasma choline concentrations has been described [17]. In both marathon runners [20,25] and cyclists [26], the absence of choline supplementation can lead to a 40% reduction in plasma choline, which might challenge cellular functions and limit performance. Evidence that con rms whether the availability of choline is also compromised during power challenges such as PT, and whether choline supplementation might bene t this sporting discipline, is scarce [17]. One study with college-aged males described supplementation with daily 600 mgalpha-glycerylphosphorylcholine and the effect on increasing lower- and upper- body isometric strength production [27]. After six days of supplementation, the athletes had signi cant gains in lower-body strength when performing isometric mid-thigh pulls on a force plate, an exercise linked to weightlifting performance. A sustainable source of choline is provided by krill oil extracted from Antarctic krill (Euphausia superba). In krill oil, choline is found in the form of phosphatidylcholine [28], which was shown to signi cantly increase plasma choline levels and some of its metabolites in a single 8 g dose plasma kinetic study in healthy volunteers [29]. A longer study over four weeks con rmed that 4.5 g daily krill oil administration signi cantly increases plasma choline and betaine concentrations in healthy young adults [30]. In an athletic setting, notably after the Ironman-distance Norseman Xtreme triathlon, serum choline concentrations signi cantly decreased by 34% from pre- to post-race [31]. On the other hand, 4 g of krill oil given for 5 weeks before the race signi cantly increased serum choline levels both before and after the race when compared to the placebo group. Krill oil, however, not only provides choline, but also n-3 PUFAs that are bound to the phospholipid molecules. In particular, the n-3 PUFAs eicosapentaenoic acid (EPA; C20:5 n-3) and docosahexaenoic acid (DHA; C22:6 n-3) have been

for 5 weeks before the race signi cantly increased serum choline levels both before and after the race when compared to the placebo group. Krill oil, however, not only provides choline, but also n-3 PUFAs that are bound to the phospholipid molecules. In particular, the n-3 PUFAs eicosapentaenoic acid (EPA; C20:5 n-3) and docosahexaenoic acid (DHA; C22:6 n-3) have been intensively studied for their immunomodulatory, anti-in ammatory and pro-resolving bene ts [32–35]. Since high-intensity training has been discussed to negatively impact immune function and in ammation [36], n-3 PUFA supplementation may provide a mean to address dietary de - ciencies and improve athletic recovery and resistance to infection [37]. Indeed, bene ts for post-exercise immune function after 2 g/d of krill oil for six weeks [38] have been demon-

Nutrients2021,13, 4237 3 of 16 strated. Moreover, EPA and DHA are known to stimulate muscle protein anabolism [39] and 3 g of krill oil given for eight weeks to resistance-trained subjects was shown to activate mTOR signaling, which is known to trigger an increase in muscle mass [40]. A mean to test EPA and DHA levels is given in the Omega-3 Index and standardization of the analytical procedure is ensured for the HS-Omega-3 Index [41]. The HS-Omega-3 Index is de ned by the EPA and DHA concentrations as a percentage of all red blood cell (RBC) fatty acids (FAs), which has been suggested to best represent long-term n-3 PUFA intake and general n-3 tissue status, in particular atrial levels [42]. EPA and DHA become biologically important once they are integrated into membranes, hence the analysis of RBC membranes provides a convenient mean to assess tissue distribution. Most importantly, the HS-Omega-3 Index has been recognized as a risk factor for sudden cardiac death and an increased risk for cardiovascular disease has been described when the HS-Omega-3 Index is 4%, an intermediate risk from >4% to <8% and a low risk if 8% [34]. It is therefore recommended to reach a target range of 8–11% to lower the risk for disease [43]. This can be achieved by consuming fatty sh or marine n-3 dietary supplements that provide long- chain n-3 PUFAs [44,45], since conversion rates from the short-chain n-3 alpha-linolenic acid (ALA) to EPA and DHA in humans are rather poor (8–20% for ALA to EPA; 0.5–9% for ALA to DHA) [46]. Besides their anti-in ammatory effects, n-3 PUFAs are also known to have anti-oxidant properties that might deal with the reactive oxygen species (ROS) that are generated pro- portionally to the intensity of the physical activity [47,48]. This exercise-induced oxidative stress can contribute to acute muscle fatigue [49], but at the same time is important for cellular signaling to adapt to training and induce an antioxidant defense response [50]. The antioxidant defense system in the body includes both endogenous enzymatic and non-enzymatic antioxidant defenses, as well as endogenous antioxidants supplied by

intensity of the physical activity [47,48]. This exercise-induced oxidative stress can contribute to acute muscle fatigue [49], but at the same time is important for cellular signaling to adapt to training and induce an antioxidant defense response [50]. The antioxidant defense system in the body includes both endogenous enzymatic and non-enzymatic antioxidant defenses, as well as endogenous antioxidants supplied by the diet. Exercise duration, intensity, tness condition and nutritional status of the athlete will de ne if the level of ROS produced is helpful or harmful [51]. Chronic exposure of high ROS levels can be harmful, exhaust the antioxidant defense systems and result in oxidative damage and impaired cellular function. Nutritional interventions can therefore be of interest to reduce oxidative stress, decrease muscle soreness and improve sports performance. So far it has been shown that 1 g/d of krill oil supplementation for six weeks reduced oxidative stress in professional rowers submitted to exhaustive exercise [52]. In a study with coronary heart disease patients that received 2 g/d krill oil for three months, it was proposed that antioxidant capacities were increased via the Kelch-like ECH-associated protein 1-NF-E2-related factor 2 (KEAP1-NRF2) signaling pathway [53]. In addition to n-3 PUFAs, antioxidant help might also be found in the form of the carotenoid pigment astaxanthin (3,3 0 -dihydroxy- , 0-carotene-4,4 0 -dione) that krill oil con- tains with both hydroxyl groups esteri ed to FA [40]. Astaxanthin has thirteen conjugated double bonds and because of their arrangement, astaxanthin has strong antioxidant prop- erties [54]. The astaxanthin dose that 2.5 g of krill oil provides is around 1.7 mg, which is below the recommended dose of 4 mg for athletes that is linked to improved muscle dam- age, time trial performance and power output [55–57]. Nevertheless, it has been suggested that the phospholipids of krill oil may increase intestinal absorption of astaxanthin [40], thereby optimizing its availability to the body for integration into cell membranes and ght against excessive free radical production in athletes [53]. As a result, this 12-week study intended to assist in laying the foundation for op- timal performance and recovery of

Nevertheless, it has been suggested that the phospholipids of krill oil may increase intestinal absorption of astaxanthin [40], thereby optimizing its availability to the body for integration into cell membranes and ght against excessive free radical production in athletes [53]. As a result, this 12-week study intended to assist in laying the foundation for op- timal performance and recovery of PT athletes by demonstrating the effect of krill oil onHS-Omega-3Index levels and plasma choline recovery after exercise, as well as on scavenging free radicals after high-power physical workouts.

Nutrients2021,13, 4237 4 of 16 2. Materials and Methods 2.1. Participants and Procedures Out of 95 screened subjects, 36 individuals tted the inclusion criteria and agreed to take hard gelatin capsules (Licap ® encapsulation) at the prescribed dose for 12 weeks prior to a training session. The nutritional composition of the study products is given in Table. Table 1.Characteristics and nutritional composition of study products. Product Krill Oil Placebo Product Name NKO TM krill oil Virgin olive oil Manufacturer Aker BioMarine, Norway Lonza Group, Switzerland Amount per capsule (mg) 500 500 Capsules per day 5 5 Weeks of administration 12 12 EPA/DHA (g/100 g) 16/6 <1/<1 Total phospholipids (g/100 g) 46 <1 Choline (g/100 g) 6 <1 Esteri ed astaxanthin (mg/kg) 691 <1 One subject was non-compliant (showing a decrease in the HS-Omega-3 Index after 12 weeks of krill oil supplementation) and therefore was excluded, leaving a total of 35 subjects(27 males and 8 females) in the nal sample. A total of 19 participants were included in the krill oil and 16 participants were included in the placebo group (Table). Table 2.Flowchart of study design. Time Phase ActivityNutrients 2021, 13, x FOR PEER REVIEW 4 of 16 2. Materials and Methods 2.1. Participants and Procedures Out of 95 screened subjects, 36 individuals fitted the inclusion criteria and agreed to take hard gelatin capsules (Licap ® encapsulation) at the prescribed dose for 12 weeks prior to a training session. The nutritional composition of the study products is given in Table 1. Table 1. Characteristics and nutritional composition of study products. Product Krill Oil Placebo Product Name NKO TM krill oil Virgin olive oil Manufacturer Aker BioMarine, Norway Lonza Group, Switzerland Amount per capsule (mg) 500 500 Capsules per day 5 5 Weeks of administration 12 12 EPA/DHA (g/100 g) 16/6 <1/<1 Total phospholipids (g/100 g) 46 <1 Choline (g/100 g) 6 <1 Esterified astaxanthin (mg/kg) 691 <1 One subject was non-compliant (showing a decrease in the HS-Omega-3 Index after 12 weeks of krill oil supplementation) and therefore was excluded, leaving a total of 35 subjects (27 males and 8 females) in

Weeks of administration 12 12 EPA/DHA (g/100 g) 16/6 <1/<1 Total phospholipids (g/100 g) 46 <1 Choline (g/100 g) 6 <1 Esterified astaxanthin (mg/kg) 691 <1 One subject was non-compliant (showing a decrease in the HS-Omega-3 Index after 12 weeks of krill oil supplementation) and therefore was excluded, leaving a total of 35 subjects (27 males and 8 females) in the final sample. A total of 19 participants were included in the krill oil and 16 participants were included in the placebo group (Table 2). Table 2. Flowchart of study design. Time Phase Activity 3 months Subject selection and characterization 1. Subject selection according to eligibility criteria 2. Subject characterization (medical examination, ECG, max exercise test, anthropometry, standard blood analysis) 15 days Group determination 1 day First training day 3. Blood analysis: Omega-3 Index, TAC, inflammatory parameters 4. Power training session (Heart rate, lactate, RPE) 5. Blood analysis: TAC, inflammatory parameters 12 weeks Supplementation period: Placebo (n = 16) / Krill oil (n = 19) 1 day Second training day 3. Blood analysis: Omega-3 Index, TAC, inflammatory parameters 4. Power training session (Heart rate, lactate, RPE) 5. Blood analysis: TAC, inflammatory parameters 2.2. Eligibility Criteria and Follow-Up Eligibility criteria were based on selecting healthy athletes of both sexes, between the ages of 21–40 who had a HS-Omega-3 Index of less than 6% and were in good health, without chronic or acute inflammatory pathology and normal blood parameters. Volunteers were excluded when they had had a muscle injury within the last 6 months or used any sort of medication. Moreover, the athletes had to be practicing the sporting discipline of PT (or any similar activity such as CrossFit™) in a sports gym with a specialized instructor for at least 3 years in a row at least four days per week. Another inclusion criterion was the commitment of the participants to avoid the use of any nutritional supplements and stay on their normal diet during the study and 15 days prior 3 months Subject selection and characterization 1. Subject selection according to eligibility criteria 2. Subject characterization (medical examination, ECG, max exercise

in a row at least four days per week. Another inclusion criterion was the commitment of the participants to avoid the use of any nutritional supplements and stay on their normal diet during the study and 15 days prior 3 months Subject selection and characterization 1. Subject selection according to eligibility criteria 2. Subject characterization (medical examination, ECG, max exercise test, anthropometry, standard blood analysis) 15 days Group determination 1 day First training day 3. Blood analysis:Omega-3 Index, TAC, in ammatory parameters 4. Power training session (Heart rate, lactate, RPE) 5. Blood analysis: TAC, in ammatory parameters 12 weeks Supplementation period: Placebo ( n= 16) / Krill oil (n= 19) 1 day Second training day 3. Blood analysis:Omega-3 Index, TAC, in ammatory parameters 4. Power training session (Heart rate, lactate, RPE) 5. Blood analysis: TAC, in ammatory parameters 2.2. Eligibility Criteria and Follow-Up Eligibility criteria were based on selecting healthy athletes of both sexes, between the ages of 21–40 who had a HS-Omega-3 Index of less than 6% and were in good health, without chronic or acute in ammatory pathology and normal blood parameters. Volunteers were excluded when they had had a muscle injury within the last 6 months or used any sort of medication. Moreover, the athletes had to be practicing the sporting discipline of PT (or any similar activity such as CrossFit™) in a sports gym with a specialized instructor for at least 3 years in a row at least four days per week. Another inclusion criterion was the commitment of the participants to avoid the use of any nutritional supplements and stay on their normal diet during the study and 15 days prior to study start. To this end, various clubs were asked for volunteers to participate. A total of 95 medical examinations were carried out with cardiometabolic-monitored maximal exercise stress tests, blood standard analyses and determination of the HS-Omega-3 index in order to reach the number considered appropriate for this pilot study (n= 40).

The study investigates the effects of krill oil on athletic performance and recovery.