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    Biosci. Biotechnol. Biochem, & (9), 1970-1976, 2001 Ia Amino Acid Supplementation Affects Hematological and Biochemical Parameters in Elite Rugby Players Masaru Ourani,!" Kimiaki Maruyama,? Masaaki Sucrra,? and Kando Kopayasut! 'The Department of Life Sciences (Sports Sciences), Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153-8902, Japan {Department of Life Science, Meiji University, Kawasaki 214-8571, Japan “Department of Health and Physical Education, Mie University, Tsu 514-8507, Japan Received December 22, 2000; Accepted May 17, 2001 Individual amino acid supplementation affects vari performance. However, This study evaluated an amino containing 1-leucine, t-isoleucine, 1-val and L-glutamine (o 3.6 g of total amino acids per dose. ‘Twenty-three rugby players were given 3.6 g, twice, dal ly of the amino acid mixture for 90 days (June-August 1994) and blood samples were collected for analyses in September 1993, March 1994, September 1994, and Sep- tember 1995. After 90 days of supplementation, almost all of the athletes reported improvement in earlier recovery from fatigue. Significant increases (P<0.05) were observed in hemoglobin, RBC count, hematocrit, and serum iron by amino acid supplementa- tion. Significant increases (P'<0.05) were also noted in {otal cholesterol and low-density lipoprotein along with decreased (P<0.05) alkaline phosphatase. AI values reverted {0 original levels when measured after one year of continued training without supplementation, Key words: amino acids; exercise; hematology; blood chemistry; sports medicine A great deal of research has been done to identify ergogenic substances most effective in improving at- hiletic performance, often focusing on the eneray re- quirements in a variety of sports and the control of food intake. Energy intake of athletes is generally in- creased or unchanged in response to exe however, it is often below the energy expenditure." Nutritional ergogenic aids are classified into four categories: substances {0 enhance anabolism and to build a favorable body composition (e.g. amino acids), substances to be readily used as an energy source (e.g. carbohydrate), substances to enhance the recovery [rom physical exhaustion (e.g. antioxi- dants), and substances playing an important role in exercise physiology (e.g. vitamins and sodium bicar- bbonate).? Amino acids are important building blocks for muscle, and at the same time, serves as an energy source for skeletal muscles. Branched chain amino acids (BCAA) are metabolized not only in liver but also in extra-hepatic tissues, such as skeletal muscles, to yield a-keto acids for gluconeogenesis. During en- durance exercise, the BCAA. pool is replenished through muscle protein catabolism. However, it is often the case that the oxidation of BCAA in skeletal muscles exceeds the catabolic capacity in prolonged ‘endurance exercise. When the BCAA are depleted, the concentration of tryptophan, relative to the BCAA, is excessively elevated in the muscle free-ami- no acid pool. As a consequence, tryptophan is chan- neled to the formation of serotonin, producing the perception of tiredness, Fatigue, and the loss of mus- cle power output ‘Arginine stimulates the secretion of various hor- ‘mones that have important actions during exercise.*” In addition, it acts in the removal of ammonia through the urea cycle to relieve the CNS fatigue Nitric oxide, derived from arginine is a potent vasodilator" to facilitate athletic performance by way of hypervolemia!” The concentration of free glutamine is approxi- mately 20 mmoles/I, accounting for 60% of the total free amino acid in skeletal muscle™” and has a nega- tive arteriovenous difference across muscles, particu- larly pronounced after prolonged exercise." In slow- twitch muscles, the intracellular concentration of ‘elutamine was 3-fold higher than in fast-twitch mus- cle," suggesting a higher demand for glutamine in endurance training. In prolonged and high-intensity exercise, plasma glutamine is increased during the ex- ercise and then decreased during the post-exercise recovery period." Glutamine is implicated in acido- sis and immunocompetence, particularly in over- training. Few reports are available on the long-term ad ministration of BCAA, arginine, and glutamine as an * To whom correspondence should be addressed: Fax 81-3$250-8273; E-mail: island87@aol.com Amino Acid Supplementation for Rugby Training wn ergogenic aid. When branched-chain amino acids were administered in high-altiuude trekking for 21 days, the muscle loss during chronic hypobaric hypoxia was prevented. In six-week endurance training on a Monarch cycle ergometer, BCAA in- creased the distribution of slow-twitch muscle fibers in elderly subjects.®” When a low dose of BCAA was administered in the cycling training, only faster adap- tation in muscle buffer capacity (4 lactate/ pH) was seen. The arginine administration in the weight training for $ weeks resulted in a decrease in body ‘mass and body fat," and increases in total strength and lean body mass and a decrease in urinary hydrox- proline excretion. In the treadmill exercise, ar nine increased maximal oxygen consumption and decreased plasma lactate.” However, in a marathon race, the creatine phosphokinase (CPK) activity was significantly elevated immediately after the race and further at 48 hours post-race. Although the post-race rise in the CPK activity was 15-fold by the arginine administration for 14 days before the race and 40- fold by the placebo, there was no statistical difference due to high variability. The arginine effec, via its metabolism (0 nitric oxide, on the prevention of in- testinal ischemic injury was inconclusive.” ‘A single dose of glutamine was effective in prevent- ing post-race infections in 200 runners.” However, ina subsequent study, the effect of glutamine on lym- phocyte distribution in a marathon race was incon- elusive.” In our previous study, the one-month oral ad- ministration of an amino acid mixture, thrice daily, to middle- and long-distance runners produced an in- crease in fasting glucose and a decrease in the CPK activity. The amino acid mixture contained 0.33 g of Lleucine, 0.264 g of isoleucine, 0.22 g of L-valine, 0.3748 of L-arginine and 0.394 g of L-glutamine in the total of 2.2 g amino acids.” In this study, the amount of amino acids was in- creased, the duration of the treatment was increased, and the type of physical training was changed from ‘endurance to intensive training to evaluate physiolog- ical actions of amino acids in relation (0 athletic per- formance. The amino acid mixture contained nine es- sential amino acids: t-leucine, t-isoleucine, L-valine, histidine, L-lysine, L-methionine, t-tryptophan, - phenylalanine and t-threonine, and three non-essen- tial amino acids: L-arginine, -glutamine and L-pro- line in the total of 3.6 g to augment the amino acid supply for protein synthesis since during the resistance training, increased muscle protein synthe- sis was linked to increased intramuscular availability of amino acids." The amino acid mixture was given to young rugby players in intensive training, twice daily, for three months. At the end of the treatment, the self-assessment of their physical condition, and the determination of hematological and biochemical parameters were made. The amounts of t-leucine, 1 isoleucine, 1-valine, L-arginine, and L-glutamine in the amino acid mixture were increased to 0.54 8, 0.43.2, 0.368, 0.65 g, and 0.61 g, respectively to potentiate specialized functions of these amino acids. Materials and Methods Experimental subjects and amino acid mixture. Twenty three members of a rugby team, which won the championship in the Japan Football League for seven years in @ row, participated in this study. The average age, height, body weight, and body fat per- centage (mean-+SE) were 27.20.4 years, 177.8 1.6 cm, 93.62.8 kg, and 15.2+0.7%, respectively ‘The experimental protocol was reviewed by the ethies committee of Shinko Hospital before the experiment for the safety of human subjects and the informed consent forms signed by the participants were filed. Test subjects had maintained a regular training schedule as with their teammates before, during, and after the 90-day administration of an amino acid ‘mixture. None of them had taken amino acid supple- ments in any forms previously. The amino acid mixture in this study contained a total of 3.6 g of nine essential amino acids: t-leucine, Lisoleucine, L-valine, L-histidine, L-lysine, t-methio- nine, L-tryptophan, L-phenylalanine and t-threonine, and three non-essential amino acids: L-arginine, t- glutamine, and t-proline. The major components of the amino acid mixture were 0.54 g t-leucine, 0.43 g L-isoleucine, 0.36 g L-valine, 0.65 g t-glutamine, and 0.61 g L-arginine. The amino acid mixture was added with carbohydrates, vitamins, sweetener and flavors to the total weight of 4.5 g and made into a one-dose packet. “The subjects were instructed to take one packet af- ter morning and evening meals, thus ingesting 7.2 ¢ of the amino acid mixture a day for 90 days. The iso- nitrogenous adjustment was deemed unnecessary be- cause the dose was merely a supplement to the regu- lar meal and 8-9% of daily protein intake, assuming 60kg body weight and 1.2-1.4 g/kg BW protein intake. Furthermore, considering 2 massive flux of ‘amino acids from recycling in protein turnover,” the difference in the daily nitrogen intake due to the treatment was negligible in the metabolic size of the nitrogen pool. Experimental protocol. With nominal disturbance to the regular training schedule of the rugby team, the administration of the amino acid mixture started on June 1, 1994 and continued through August for 90 days. Blood sampling was done before and after the 90-day test period: September 1993, March 1994, ‘September 1994, and September 1995, The survey of physical feeling was done at the midway and at the end of the 90-day test period. ws M. Ouran et a Blood sampling and blood analyses. Blood sam- ples were collected from the antecubital vein after overnight fasting. No unscheduled, intensive training was done the night before the blood sampling. A. 10-ml blood sample was collected in an uncoated vacuum tube and immediately centrifuged for biochemical analyses and a 2-ml aliquot was collected in a tube coated with EDTA-K; for the complete blood count. The complete blood count included white blood cell count (WBC), red blood cell count (RBC), hematocrit (Ht), and hemoglobin (Hb). The counting was made using a Coulter-STKA auto-analyzer. Biochemical parameters were high density lipoprotein (HDL), low density lipoprotein (HDL), blood urea nitrogen (BUN), glutamate-oxalacetate transaminase (GOT), glutamate-pyruvate trans- aminase (GPT), y-GTP (gamma-glutamyl transpepti- dase), creatinine, alkaline phosphatase (ALP), total cholesterol (TC), triglyceride (TG), total protein (TP), and serum iron (Fe). Blood biochemistry was. analyzed with an auto-analyzer (Hitachi 7359) with an exception of LDL. The concentration of LDL was. measured by the method of Friedelwald er al. (1972). Self-assessment of subjective, athletic factors. For- ty five days into the twice daily ingestion of the amino acid mixture and at the end of the 90-day test period, experimental subjects were interviewed and asked the following questions: changes in vigor, appetite, and general physical condition, and changes in the rate of recovery from training fatigue: lower back pain, and muscle ache. The answer was limited to either “'yes”” or no” Statistical analysis. The paited £ test was used for the comparison of two means when the observations in September 1994 were compared with the observa- tions preceding the administration of the amino acid mixture: March 1994 and September 1993, and the observations long after the termination of the ad- ministration of the amino acid mixture: September 1995. The observations in March 1994 served as their own control and effects of the 90-day daily dose of 7.28 of amino acids were reflected in the observa- tions in September 1994. The criterion for statistical significance was set at the probability of 0.05 (P-<0.08). Significant differences from the values ob- served in September 1994 are indicated with an aster- isk (*). Results In the physical feeling survey, 14 of 23 study par- ticipants perceived increased vigor and 12 of 23 par- ticipants concluded in their self-assessment an im- provement in the general physical condition and Quicker recovery from the training fatigue in 45 days Of the amino acid intake. After 90 days, 20 of 23 study participants perceived an increase in vigor, and 22 participants reported an improvement in the general physical condition. Eight of 23 participants noted an increase in appetite, faster recovery from the training muscle fatigue, and the reduced lower back pain. Body weight, complete blood cell counts, biochem- ical parameters, and body fat in March 1994, Sep- tember 1994, and September 1995 are summarized in Table I. The body weight stayed constant throughout the study. When the values before (March 1994) and at the end (September 1994) of the 90-day adminis- tration of the amino acid mixture were compared, hematocrit, hemoglobin, iron, total cholesterol, and low-density lipoprotein were elevated (P<0.05) at the end of the amino acid administration. On the other hand, alkaline -phosphatase activity was reduced (P-<0.08) at the end of the treatment. ‘When the values one year after the termination of the treatment (September 1995) were compared against the values at the end of the 90-day adminis- tration of the amino acid mixture (September 1994), RBC count, hematocrit, hemoglobin, and total cholesterol were reduced (P-<0.05), whereas blood urea nitrogen, creatinine, 7-GTP, and alkaline phos- phatase activity were elevated (P'<0.05). ‘The hematocrit value, the RBC count, the hemoglobin concentration, and the iron concentra- tion were plotted against the time in Fig. 1. The ob- servations in September 1993 were added as reference values. In all cases, values were elevated by the 90-day administration of the amino acid mixture and returned to the pre-treatment level in one year (P<0.05). Total cholesterol, triglyceride, high-density lipoprotein and low-density lipoprotein concentra- tions were plotted against time in Fig 2. Low-density lipoprotein and total cholesterol concentrations rose after the treatment and declined thereafter (P<0.05). On the other hand, high-density lipoprotein remained constant before, immediately after, and a year after the treatment, and alkaline phosphatase were depressed after the treatment and then, restored (0 the pre-treatment level (P<0.05). For alkaline phosphatase, see Table I (not included in Fig. 2), Discussion ‘The self-assessment of the rugby players who par- ticipated in the study revealed the increased vigor and faster recovery from muscle fatigue during the daily routine of the rugby training when they received ‘owice daily doses of the amino acid mixture, totaling 7.2. of the amino acid mixture which contained leu- cine, isoleucine, valine, glutamine, and arginine as Amino Acid Supplementation for Rusby Training 3 Changes in Body Weight, BUN, Creatine, Hematopoetic Parameters, Biochemistry and Enzymes inthe Blood of Rugby Footbal, Players before, Immediately alter, and One Year ater the 90-day Adminstration of the Amino Aci Mixture Table 1. (a) 1994 March Body weight 93.6228 BUN’ 164208 Creatine Lo9:=0.08 wae REC Hematocrit Henoslabin tron ‘Total protein ‘Total cholesterol, High density lipoprotein Low density lipoprotein Trieyoe Ms 18 cor a4 Ger’ m2 poTP 2622 Alkaline phosphatase 72208 = An anerisk indicates 9 inant diference (P<) 2 Blood urea mizogen. "White blood cll cout. the primary components, for 90 days. Some par- ticipants also noted increased appetite and reduced lower back pain ‘The 90-day administration of the amino acid mix- ture elevated the hematocrit value, the RBC count, and hemoglobin and iron concentrations (Fig. 1), suggesting that the production of blood cells, namely red blood cells, was augmented and the capacity 10 carry the oxygen in the blood stream was augmented. Ithas been reported that blood volume varied with changes in ambient temperature and the amount of exercise." Since rugby players maintained their ‘raining at a constant pace through the year, any im- plication of the exercise intensity to the blood volume was ruled out, The blood concentration is likely to ‘occur more often in summer. In this study, the ex- periment started in June and continued through Au- gust and the hematocrit value was indeed significant- ly increased. In order to resolve the confounding effect of the seasonal fluctuation in blood volume, the hematocrit values, the RBC count, and hemoglobin and iron concentrations in September 1994 were compared to the measurements in September 1993 and September 1995. While there was no differences jin the hematocrit value, the RBC count, and hemoglobin and iron concentrations between Sep- tember 1993 and September 1995, the measurements in September 1994 were significantly higher than the measurements in September 1993 and/or September 1995, thus concluding that the enhanced hematopoie- sis was the result of the 90-day administration of the ‘Sampling Time! Statist (8) 1994 (©) 1995 September September (AY 5 (B)—— (BY VSO) osse27 93.2226 16.1206 17208 0 1104003 1,140.02 . 40200 6500300 0 S162 13 39026 2 462408 : 15.602 : ine? : 72201 ies : 2 S222 10910 : 15012 2622 asd awed : sae03 : : “The 90a aiinistaton ofthe amine acid inte was dane fom June through AugU, 199, * Gluamiconlesis transaminase.” Guam pyruic wansuminase amino acid mixture. ‘One of clinical symptoms of liver cirrhosis is ane- mia. When an amino acid mixture containing 2.04 ¢ of leucine, 1.93 g of isoleucine, 1.6 g of valine, and 0.94 g of other amino acids were given to liver cirrho- sis patients twice or thrice a day for 1 to 2 months, the hematocrit value, the RBC count, the hemoglobin concentration were significantly i creased in 8 weeks of the treatment and remained elevated through the end of the study." Hypercholesterolemia in rabbits was induced by dietary casein protein.” Elevated levels of cholesterol and low-density lipoprotein in the blood were obtained by feeding the casein amino acid mix- ture,” more precisely by feeding lysine, leucine, and methionine." In this study, the oral administra- tion of the amino acid mixture resulted in elevated total cholesterol and low-density lipoprotein in the blood. However, the normal range for total cholesterol is in the range of 150 to 220 mg/dl,” and the values observed in this study were well within the range. Alkaline phosphatase, GOT, GPT, and y-GTP are liver function parameters and the rise in their activi- ties indicates disease conditions and the disturbance to the liver function. The normal range of alkaline phosphatase activity is 3 to 10 KAU" and reduced alkaline phosphatase activity due to the administra- tion of the amino acid mixture was favorable. In liver cirrhosis patients, the oral administration of an ami- no acid mixture consisting primarily of branched 1974 M. Ouran tal tenance af le de |e 5 a i a = ve | ” 3 7 soo} | =| ve 1450) " 90! Fig. 1. Changes in Blood Components Related to Oxygen-cat- ‘ying Capacity: Hematocrit Value, Red Blood Cell Count, Hemoglobin, and Iron, One Year before, Six Months before, Immediately afer, and One Year after the 90-

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