Dietary Intake of Protein and Essential Amino Acids for Sustainable Muscle Development in Elite Male Athletes

letes need to develop a relatively high muscle mass and low body adipose tissue for the sake of better athletic performance. A full range of nine essential amino acids and eleven non-essential amino acids have to attend in appropriate amounts for protein biosynthesis. The aim of the observational comparative cross-sectional study was to assess the association between the diet quality pro le and training-induced muscle mass estimated by bioelectrical impedance among elite male athletes. The research sample comprised 18.1 3.1 year-old Lithuanian professional male athletes (n = 234). The study participants were enrolled to complete 24-h dietary recalls of three non-consecutive days. The body composition was assessed using the bioelectrical impedance analysis (BIA) method. The present study showed a signi cant insuf ciency of the mean carbohydrate intake of 5.7 g/kg/day in a group of aerobic male athletes. The lower muscle mass of aerobic male athletes was related to the lower-carbohydrate diet (adjusted odd ratio (OR adj) 0.3; 95% con dence interval (CI): 0.1–0.7). The mean protein intake of 1.8 g/kg/day was optimal for anabolism in the samples of both anaerobic and aerobic male athletes. The protein intake in appropriate doses was potentially associated

of aerobic male athletes. The lower muscle mass of aerobic male athletes was related to the lower-carbohydrate diet (adjusted odd ratio (OR adj) 0.3; 95% con dence interval (CI): 0.1–0.7). The mean protein intake of 1.8 g/kg/day was optimal for anabolism in the samples of both anaerobic and aerobic male athletes. The protein intake in appropriate doses was potentially associated with an increase in muscle mass only in anaerobic male athletes (OR adj2.2; 95% CI: 1.3–3.7). The positive relationship was revealed between the possible muscle mass gain and the increased intakes of amino acids such as isoleucine and histidine among anaerobic athletes (OR adj2.9; 95% CI: 1.1–4.7 and OR adj2.9; 95% CI: 1.0–4.3, respectively). An inverse feasible association was indicated between a higher intake of valine and lower muscle mass quantities among anaerobic male athletes (OR adj0.1; 95% CI: 0.1–0.5). The recommendations for sports nutritionists should emphasize the necessity of advising professional athletes on dietary strategies on how to manipulate dietary amino acid composition with respect to achieving long-term body composition goals. Keywords: nutrition; diet; elite athletes; anaerobic sports; aerobic sports; body composition; muscle development; protein; essential amino acids 1. Introduction Nutrition is characterized as a certain behavior by which nutrients are consumed in suf cient amounts for ensuring a good standard of living and maintaining a healthy lifestyle. The daily nutritional requirements for athletes are particularly strict. More speci cally, athletes must consume nutrients in increased and balanced quantities. In addition, the nutritional pro le of athletes is important for optimizing athletic performance and depends on factors, namely sex, age, branch of sports, and athletic goals, as they appear to be related to body composition [1]. Athletes need to develop a relatively high muscle mass and low body adipose tissue for the sake of better athletic performance. The striated muscle tissue is the most metabolically active bodily tissue. Muscle proteins are unstable, as they have a permanent ability to turn over, i.e., degrade and synthesize. The changes in both the synthesis and degradation of muscle proteins play an important role in the Nutrients2023,15, 4003.

tissue for the sake of better athletic performance. The striated muscle tissue is the most metabolically active bodily tissue. Muscle proteins are unstable, as they have a permanent ability to turn over, i.e., degrade and synthesize. The changes in both the synthesis and degradation of muscle proteins play an important role in the Nutrients2023,15, 4003.

Nutrients2023,15, 4003 2 of 18 recovery and remodeling of muscle proteins following physical loading. The changes in muscle mass depend on the changes in the protein content in myo bril, accordingly. Thus, such metabolic regulation has a key role in the adaptations of skeletal muscle (in terms of size) to exercise training. Approximately 5% of essential amino acids (EAAs) from protein catabolism are oxidized in muscles at partial levels and inaccessible for a new protein translation. In all, 25% of EAAs released into the blood are used by other body tissues. The remaining amount (70%) of essential amino acids is recycled into protein synthesis [2]. Thus, protein degradation rates always surpass the biological process of the synthesis of new protein cells by 30% on average, depending on an insuf cient EAA intake. The higher rate of protein synthesis can only occur with protein intake in appropriate doses [3]. It has been found that, due to aminoacidemia, the positive muscle protein net balance can be ensured between 1.5 and 3 h after consumption of a high-protein meal within 30 to 45 min during the postprandial state [4]. In addition, the amino acid-mediated anabolic stimulation of muscle protein synthesis relies on the amount of dietary protein and amino acid intake, i.e., the biological value of proteins. There are a total of 20 amino acids that make up muscle proteins. Nine out of these 20 amino acids belong to the group of EAAs. EAAs cannot be produced by the human body in physiologically signi cant quantities, and, therefore, are necessarily obtained through an adequate diet. The remaining 11 amino acids are categorized as non-EEAs, as they might be produced by the physical body [5,6]. Thus, the full range of nine EAAs and the 11 non-essential amino acids should be available in appropriate amounts for protein biosynthesis [7]. Hence, the creation of new proteins may be restricted by a limited presence of any EAA, whereas the lack of non-EAAs could be offset by the boosted de novo synthesis [8]. On the other hand, an essential variable that can be related to the general ef

11 non-essential amino acids should be available in appropriate amounts for protein biosynthesis [7]. Hence, the creation of new proteins may be restricted by a limited presence of any EAA, whereas the lack of non-EAAs could be offset by the boosted de novo synthesis [8]. On the other hand, an essential variable that can be related to the general ef cacy of protein/amino acids in the course of exercise training is the absolute protein intake per day. The factors associated with protein timing and the quality of protein used are equally relevant to assessing the effects of dietary protein intake during long-lasting exer- cise participation [9]. Therefore, a large number of meta-analyses and experimental and observational studies without intervention have been conducted to summarize the effects of the consumption of protein supplements on the occurrence of changes in body composition, muscular power and strength or the levels of bodily adaptation to exercise [10–20]. However, the research has focused on protein supplementation in the physically active adult population, whereas the diet quality, nutrient intake, and amino acid composition in the subjects' diets were uncontrolled. Scienti c attention should also focus on the nutritional pro le when athletes lack energy/carbohydrates and protein in their diet, as both the total daily protein intake and the quality of protein intake can be useful for the increase in and growth of muscle cells during exercise training. This scienti c theory is supported by meta-analysis, which explains that the total protein consumption, with the modern Western diet, is a more important factor than the daily distribution of protein intake in triggering muscle hypertrophy during physical training [15]. Therefore, further determination of the nutritional status and body composition in athletes remains necessary, as no speci c agreement has been reached on the results of protein intake to boost muscle hypertrophy. Additionally, so far there have been no studies aiming to summarize the association between the muscle mass (in terms of size) of elite athletes and the nutrient intake in most countries of the European Union and the Baltic States. The aim of this study was to assess

has been reached on the results of protein intake to boost muscle hypertrophy. Additionally, so far there have been no studies aiming to summarize the association between the muscle mass (in terms of size) of elite athletes and the nutrient intake in most countries of the European Union and the Baltic States. The aim of this study was to assess the association between the diet quality pro le and the training-induced muscle mass estimated by bioelectrical impedance among elite male athletes. 2. Materials and Methods 2.1. Data Collection and Study Participants A total of 336 Lithuanian elite athletes of the eligible population were selected from the list approved by the Lithuanian National Olympic Committee (LNOC). Strati ed random sampling was used to enroll elite male athletes for the observational study. The following

Nutrients2023,15, 4003 3 of 18 requirements were the main criteria for inclusion of athletes in the study: (1) professional male athletes participating in the preparatory training period; (2) participants in the Europe and World Athletics Championships; (3) candidates for the Olympic Team. A more detailed algorithm for the recruitment process is presented in Figure.Nutrients 2023, 15, 4003 3 of 18 2. Materials and Methods 2.1. Data Collection and Study Participants A total of 336 Lithuanian elite athletes of the eligible population were selected from the list approved by the Lithuanian National Olympic Committee (LNOC). Stratified ran- dom sampling was used to enroll elite male athletes for the observational study. The fol- lowing requirements were the main criteria for inclusion of athletes in the study: (1) pro- fessional male athletes participating in the preparatory training period; (2) participants in the Europe and World Athletics Championships; (3) candidates for the Olympic Team. A more detailed algorithm for the recruitment process is presented in Figure 1. Figure 1. Enrollment flowchart for elite male athletes. Finally, during the period from 2018 to 2019, 234 elite male athletes aged 18.1 ± 3.1 years involved in exercises for Olympic sports, namely boxing, judo, Greco-Roman wres- tling, taekwondo, weightlifting, basketball, gymnastics, high jump, rowing, road cycling, swimming, skiing, biathlon, long-distance running, and modern pentathlon, were in- cluded in the comparative cross-sectional study (Table 1). Table 1. Distribution of male athletes (in percentage) engaged in different sports according to the dominant energy-producing pathway in the body. Anaerobic Sports Eligible Analyzed Aerobic Sports Eligible Analyzed n = 138 n = 104 n = 198 n = 130 n % n % n % n % Boxing 15 10.9 14 13.5 Rowing 37 18.7 28 21.5 Judo 13 9.4 6 5.8 Road cycling 51 25.8 31 23.8 Greco-Roman wrestling 30 21.7 29 27.9 Swimming 44 22.2 29 22.3 Taekwondo 4 2.9 3 2.9 Skiing 19 9.6 12 9.2 Weightlifting 8 5.8 6 5.8 Biathlon 22 11.1 17 13.1 Basketball 53 38.4 39 37.5 Long-distance running 13 6.6 8 6.2 Gymnastics 4 2.9 2 1.9 Modern pentathlon 12 6.1 5 3.8

5.8 Road cycling 51 25.8 31 23.8 Greco-Roman wrestling 30 21.7 29 27.9 Swimming 44 22.2 29 22.3 Taekwondo 4 2.9 3 2.9 Skiing 19 9.6 12 9.2 Weightlifting 8 5.8 6 5.8 Biathlon 22 11.1 17 13.1 Basketball 53 38.4 39 37.5 Long-distance running 13 6.6 8 6.2 Gymnastics 4 2.9 2 1.9 Modern pentathlon 12 6.1 5 3.8 Disc throw, javelin throw 7 5.1 3 2.9 – – – High jump 4 2.9 2 1.9 – – – Figure 1.Enrollment owchart for elite male athletes. Finally, during the period from 2018 to 2019, 234 elite male athletes aged18.1 3.1 years involved in exercises for Olympic sports, namely boxing, judo, Greco-Roman wrestling, taekwondo, weightlifting, basketball, gymnastics, high jump, rowing, road cycling, swim- ming, skiing, biathlon, long-distance running, and modern pentathlon, were included in the comparative cross-sectional study (Table). Table 1. Distribution of male athletes (in percentage) engaged in different sports according to the dominant energy-producing pathway in the body. Anaerobic Sports Eligible Analyzed Aerobic Sports Eligible Analyzed n = 138 n = 104 n = 198 n = 130 n % n % n % n % Boxing 15 10.9 14 13.5 Rowing 37 18.7 28 21.5 Judo 13 9.4 6 5.8 Road cycling 51 25.8 31 23.8 Greco-Roman wrestling 30 21.7 29 27.9 Swimming 44 22.2 29 22.3 Taekwondo 4 2.9 3 2.9 Skiing 19 9.6 12 9.2 Weightlifting 8 5.8 6 5.8 Biathlon 22 11.1 17 13.1 Basketball 53 38.4 39 37.5 Long-distance running 13 6.6 8 6.2 Gymnastics 4 2.9 2 1.9 Modern pentathlon 12 6.1 5 3.8 Disc throw, javelin throw7 5.1 3 2.9 - - - High jump 4 2.9 2 1.9 - - -

Nutrients2023,15, 4003 4 of 18 The Lithuanian elite male athletes engaged in different sports were classi ed into two groups, namely anaerobic (75.3%, n = 104) and aerobic (65.7%, n = 130) athletes, depending on two primary energy-producing pathways in the body [21]. All tests related to the study were performed during the preparatory training period prior to the competitions in a cohort of male elite athletes. The average physical load duration in study participants matched 176.9 62.6 min per day. The physical activity levels of elite male athletes fully conformed to the intensity zones approved for training plans by the Lithuanian Sports Centre (LSC) and LNOC (Table). Table 2.Training programs for elite male athletes. Variables Anaerobic Sports (n = 104) Aerobic Sports (n = 130) Training period Special training Special training Training experience, years 8.2 3.7 7.8 3.9 Exercise per month, days 23.3 3.1 23.1 3.6 Duration of training, hours per month 41.9 12.1 48.6 17.4 Duration of training, hours per day 2.8 1.1 3.1 1.1 Physical activity levels were allocated for ve intensity zones depending on energy-producing pathway during workouts (% 1 ) Aerobic endurance training, recovery: heart rate equaled 130 10 bpm, blood lactate levels were up to 2 mmol/L 10–17% 17–44% Aerobic strength training: heart rate equaled 150 10 bpm, blood lactate levels were 2–4 mmol/L, and special muscular power increased at the anaerobic threshold 19–41% 37–70% Aerobic and anaerobic glycolytic strength training: heart rate equaled 170 10 bpm, blood lactate levels were 4–12 mmol/L 13–36% 9–34% Anaerobic glycolytic strength training: heart rate 181 bpm, blood lactate levels were up to 21 mmol/L 0–6% 0–7% Anaerobic phosphocreatine strength training: blood lactate levels were 1.5–6 mmol/L 0–2% 0–4% bpm—beats per minute; 1 —time allocated for intensity areas during workouts (%). 2.2. Anthropometric Measures The analysis of the body composition of elite athletes was conducted at the Lithuanian Sports Medicine Centre (LSMC). A stadiometer was used for measuring the height to the nearest 1 cm of athletes. Due to the bioelectrical impedance analysis (BIA) conducted as a non-invasive testing method referring to the third level of validity approach,

during workouts (%). 2.2. Anthropometric Measures The analysis of the body composition of elite athletes was conducted at the Lithuanian Sports Medicine Centre (LSMC). A stadiometer was used for measuring the height to the nearest 1 cm of athletes. Due to the bioelectrical impedance analysis (BIA) conducted as a non-invasive testing method referring to the third level of validity approach, the outcomes of which were highly correlated to those of dual-energy X-ray absorptiometry (DXA) [22,23], it was possible to analyze and estimate the body composition using the electrical resistance of various tissues of the body [24,25]. In 234 male athletes, the body composition evaluation using the BIA method via an X-scan (Kyungsan City, Republic of Korea) device was conducted. Impedance measurements were performed by using 5 different electrical signals of 5, 50, 250, 550, and 1000 kHz. The BIA provided information on the body composition of athletes: body weight (BW) (kg), fatty tissue mass (FM) (kg and percentage of BW), lean body mass (LBM) (kg and percentage of BW), muscle mass (MM) (kg and percentage of BW). The muscle and fat mass index (MFMI) of each athlete was calculated by dividing MM (kg) by BF (kg). For BIA outcomes and the assessment of individual body mass components (FM, MM, and MFMI), appropriate scales were used. These norms identi ed for elite athletes were previously invented, published, and currently used by the authors for the aims of this study [26]. 2.3. Nutritional Assessment The resting metabolic rate (RMR) was estimated using the Harris–Benedict equa- tion [27]. Twenty-four-hour physical activity recalls were gathered on the same day the athletes reported for their daily calorie consumption. The additional energy expenditure

Nutrients2023,15, 4003 5 of 18 during exercise (training energy expenditure (TEE)) was estimated in the light of the recom- mendations of the American Dietetic Association, Dietitians of Canada, and the American College of Sports Medicine [28] as well as the report of empirical research by Ainsworth and colleagues [29]. Considering the fact that there is currently no “gold standard” for assessing di- etary intake, dietary recalls, as the most common approach used in sports nutrition re- search[1,30–35] , were also applied in our study. The 24-h dietary recall method was used in the dietary assessment of athletes [28,30,36,37]. The participants were instructed to maintain their habitual diet throughout the study and nally were enrolled to complete the forms for their 24-h dietary recalls of three non-consecutive days with the assistance of a sports dietitian [38]. The forms with dietary recalls were collected from all participants by a sports dietitian using a direct personal interview method during the physical examination of athletes at the LSMC. All food and drinks consumed by the study participants were recorded by the trained interviewer in line with the basis of the amounts of food provided in the Atlas of Foodstuffs and Dishes [39]. A list of the athletes' average daily food intakes was compiled during the next data processing phase. The nutritional analysis software NutriSurvey (the English translation of a professional German nutrition software program (EBISpro)) was applied for tracking the ingredients used in food recipes in order to calculate the nutrition values of individual food items (http://www.nutrisurvey.de/ January 2018)). Additionally, the NutriSurvey function "Food/Include more foods from other databases" was used as well as the nutrition values of food items manually integrated from the Lithuanian food database [40]. Each sport discipline is based on the origin of training and is related to one of two primary energy-producing pathways in the body along with nutrient requirements. There- fore, sports nutritionists working with athletes involved in different sports (anaerobic and aerobic) have been provided with nutrition guidelines established by the international sporting committees, namely, the International Society of Sports Nutrition (ISSN), the International Olympic Committee (IOC),

the origin of training and is related to one of two primary energy-producing pathways in the body along with nutrient requirements. There- fore, sports nutritionists working with athletes involved in different sports (anaerobic and aerobic) have been provided with nutrition guidelines established by the international sporting committees, namely, the International Society of Sports Nutrition (ISSN), the International Olympic Committee (IOC), and the American College of Sports Medicine (ACSM). Consequently, the intakes of carbohydrates and protein in the athletes we studied were assessed in accordance with the recommended values provided in the these scienti c recommendations [41–46]. More speci cally, the carbohydrate content recommended for athletes is consistent with 7–10 g/kg/day. The protein intake for athletes should be within the limits of 1.4 to 2.0 g/kg/day [47,48]. Taking into account that some uncertainty remains over the adult EAA requirements, the World Health Organization (WHO) has proposed a recalculation of the individual amino acid requirements divided by the total protein requirement. In this context, the EAA requirements for athletes were assessed in accor- dance with the recommendations made by the WHO, following an empirical recalculation in accordance with the standards applicable to athletes [49,50]. Additionally, the widely used technique for nutritional assessment was applied for estimating the nitrogen balance (NB) in male athletes. The NB of all of the athletes was calculated using the formula: NB (g/N/day) = DNI UNA NUN UNPL, where (1) DNI—dietary nitrogen intake estimated as dietary protein intake (DPI)/6.25 [51]; (2) UNA—urea nitrogen appearance; (3) NUN—non-urinary nitrogen excretion (e.g., ammonia, uric acid, creatinine, amino acids) [52,53] estimated as 31 mg/kg [54]; (4) UNPL—urinary nitrogen protein losses (esti- mated as 2 g for gastrointestinal and integumentary (dermal) losses) [55]. The data were rearranged to empower the estimation of UNA from protein intake (using Bergstrom's formula, UNA = (DPI 19)/7.62) [56]. In the meantime, fat intake may uctuate between 20% and 35%, contingent on the daily caloric consumption by athletes. 2.4. Statistical Analysis The cross-sectional study was conducted in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist [57].

The study assesses the association between diet quality and muscle mass in elite male athletes.