The objective of this study is to examine if a carbohydrate-protein, carbohydrate, or placebo beverage consumed immediately and two hours following eccentric resistance exercise influences:
1. Muscle glycogen resynthesis up to 72 hr post-exercise,
2. Serum levels of hormones associated with muscle glycogen synthesis and muscle protein synthesis (insulin, testosterone, growth hormone),
3. Serum levels of a hormone associated with muscle catabolism and inflammation (cortisol),
4. A marker of skeletal muscle membrane damage (serum creatine kinase) and contractile protein breakdown (urinary 3-methylhistidine), and
5. Quadriceps muscle soreness and isokinetic quadriceps exercise performance.
The hypotheses of this study have been formulated in two parts: (a) on the day of eccentric exercise, experimental day 5, and (b) morning values before and following the eccentric resistance exercise. All hypotheses are stated in the alternate form so that direction is specified.
H1: A carbohydrate-protein beverage, consumed immediately and 2 hr after eccentric resistance exercise, may help for lower serum creatine kinase and muscle soreness immediately, 3 hr, and 6 hr after eccentric resistance exercise.
H2: A carbohydrate-protein beverage, consumed immediately and 2 hr after eccentric resistance exercise, may help in higher serum insulin, testosterone, growth hormone and glucose, immediately, 3 hr, and 6 hr after eccentric resistance exercise.
H3: A carbohydrate-protein beverage, consumed immediately and 2 hr after eccentric resistance exercise may help for higher skeletal muscle glycogen resynthesis measured 24 and 72 hr after eccentric resistance exercise.
H4: A carbohydrate-protein beverage, consumed immediately and 2 hr after eccentric resistance exercise, may help for lower morning urinary 3-methylhistidine on experimental days 6-10.
7. Operational Definitions of the Terms:
7.1 Concentric Muscular Contraction. This is a kind of dynamic muscular contraction which muscle shortening as it develops tension or force.
7.2 Eccentric Muscular Contraction. This is a kind of dynamic muscle contraction which muscle lengthening as it develops tension or force that has been shown to produce the most structural damage to the muscle and subsequent muscle protein breakdown.
7.3 One Repetition Maximum (1-RM). The maximum amount of mass that can be lifted for only one time and is considered an indicator of muscle strength.
7.4 Peak Oxygen Uptake (VO2peak). The highest level of oxygen use by skeletal muscle, heart, and lungs during an incremental cycling or running exercise protocol to exhaustion. It is considered an indicator of aerobic or cardiorespiratory physical fitness.
7.5 Isokinetic Peak Torque. The maximal amount of rotational force produced at a joint when speed of movement is held constant.
7.6 Creatine Kinase (CK). An enzyme found in skeletal muscle that is believed to “leak”out of the muscle into the bloodstream in response to damage-producing highintensity exercise, particularly eccentric muscular contractions.
7.7 3-Methylhistidine (3MH). An amino acid, which is a constituent of the actin and myosin contractile components in muscle and is believed to be an indicator of muscle protein breakdown. Since this amino acid cannot be further metabolized or incorporated into new muscle protein, it is excreted and can be detected in the urine (Ballard & Thomas, 1983; Young & Munro, 1978).
7.8 Glycogen. The storage form of carbohydrate concentrated in the heart, liver, and skeletal muscle that consists of chains of glucose linked at the 1, 4 and 1, 6 carbon atoms.
7-9 Carbohydrate-branche chain amino acid (BCAA) bevarage. The beverage that it is include of 1.0 g C/kg body wt, 1.2 g of BCAAs/kg body wt; in capsules: leucine 54%, isoleucine 19%, valine 27%, skim milk with strawberry-flavored Nestle Quik.
7-10 muscle damage. The muscle damage is the damage that process may be initiated due to mechanical or biochemical stress on the muscle fibers during novel eccentric exercise.
8. Scope and limitations:
The results of this study maybe potentially beneficial to athletes who participate in multiple workouts per day (viz., triathletes, football players), in multiple competitions in one day (wrestlers), and in competitions over several consecutive days (viz., swimmers, gymnasts). The type of post-exercise beverage consumed by these athletes may promote a hormonal environment more favorable for muscle glycogen and muscle protein synthesis as well as lessen indicators of muscle damage, breakdown, and inflammation.
The following are potential limitations of this study:
1. The results of this study may be generalized to males of similar age and training status as defined by the subject sample, not for others.
2. Although practice sessions will enhance the familiarity with exercise training which may influence their effort during the isokinetic testing, aerobic cycling, and eccentric resistance exercise.
3. The timing of blood draws will limit to fasting, immediate, 3 hr, and 6 hr postexercise on day 5. Therefore, the response of serum insulin and serum glucose to the treatment beverages is limited to these time points.
4. A dietary baseline period will be limited to four days that may affect baseline blood and urine measurements.
5. No biochemical assessments of nutritional status will be performed on the subjects and also the muscle temperature is uncontrollable.
6. The subjects will not housed on-campus for the experimental period. They will return to their homes after their evening meals and returned the following morning.
This study will be delimited as follows:
1. Subject selection will be delimited to males, age 20-30 years, who will present on the Polytechnic Institute and National University of Iran.
2. Subjects will be delimited to those subjects who are free from chronic disease or orthopedic limitations that would preclude their involvement in strenuous exercise.
3. Subjects will be untrained fitness people who were not exposed to any weight training exercise on their legs or no experience of running training within the past six months.
4. Subjects will not have a history of lactose intolerance.
5. Although the subjects will consume a controlled diet for 9 days, the carbohydrate- Carbohydrate-branche chain amino acid (BCAA), carbohydrate, and placebo treatment beverages will give only on day 5 following eccentric resistance exercise.
6. Diet will be controlled by giving them specially prepared meals in a classroom or by giving the subjects a boxed meal to consume away from the preparation area.
Twenty-seven healthy student, untrained male, between 20 and 30 years of age from universities of Iran will volunteer for the study. Subjects will be recruited based on the following criteria:
(a) Untrained fitness status with particular emphasis on no resistance training involving the legs or regular running for at least 6 months, (b) no medical contraindications such as chronic disease or orthopedic limitations, (c) no lactose intolerance or milk allergy, (d) no allergy to local anesthetics, (e) no apparent difficulties during blood draws, and (f) availability during the proposed data collection periods.
These subjects will fill up more details about their medical and exercise history so that their inclusion as sample will be confirmed. Their consent will be taken about their whole hearted cooperation and physical presence during data collection time periods. Based on criteria from the medical and exercise history form, 27 males will chose to participate in the study. Approximately one week prior to their experimental period, subjects will report to the laboratory for testing.
The experimental period will last only for 9 days plus the morning of day 10. An outline of the study design is shown in Figure 1.
Subjects will consume a controlled diet for 9 days, and the first 4 days will be considered as a baseline period where body weight will be measured and energy intake will be adjusted in order to maintain body weight. Twenty-four hour urine collections will complete on day 3-9. Fasting blood samples will be obtained from the antecubital vein on the mornings of days 4-10. On the evening of day 4, the subjects will report to the laboratory to perform 40 min of stationary cycling at 70% of peak VO2. Heart rate and rhythm will be continuously monitored by telemetry, and heart rate and RPE will record at 5 min intervals. A rest period of up to 2 min will be allowed if the subjects become fatigue during the cycling. This endurance exercise will follow by 5 bouts of 1-min maximal cycling sprints interspersed with 2 min of light pedaling. Following the exercise bout, the subjects will be given a low-carbohydrate snack (290 kcal, 10% carbohydrate, 21% protein, 69% fat). The rationale for this snack will to keep the subjects’ glycogen levels low until they perform the eccentric resistance exercise the following morning.
On the morning of day 5, the subjects will report to the laboratory to perform 10 sets of 10 repetitions of isotonic eccentric quadriceps contractions with their dominant leg using the same leg on extension machine on which the 1-RM was previously determined. Resistance will set at 120% of 1-RM. Research assistants will raise the weight stack while the subject will ask to lower the weight for a 4 s count. Maintenance of this count becomes more difficult for the subjects as the sets progress. One minute rest will be given between each set. Additional blood samples will be taken immediately (IPE), 3 h (3 h PE) and 6 h (6 h PE) post exercise.
Immediately after the eccentric resistance exercise and blood sampling are over, the subjects will undergo a needle muscle biopsy from the vastus lateralis of the exercised leg using the suction technique (17). Additional muscle biopsies will be taken 24 h (24 h PE) and 72 h (72 h PE) post eccentric exercise. A new incision will make approximately 3 cm away from the previous incision for subsequent biopsy samples (10). The weight of the biopsies will be taken quickly, to be kept in liquid nitrogen, and will be stored at -80oC until analysis. Muscle glycogen will be assessed spectrophotometrically using the method of Lo et al. (26).
Following the muscle biopsy procedure and 2 h later, the subjects will be randomly assigned to receive either C (1.0 g C/kg, Gatorade, Chicago, IL), CP (1.0 g C/kg, 1.2 g BCAA/kg, skim milk with strawberry-flavored Nestle Quik, (Nestle Corp., San Francisco, CA), or aspartame-flavored placebo (P) (Crystal Light, Kraft Foods, Inc., White Plains, NY). Both C and CP will make as 13% solutions.
Quadriceps muscular soreness will be assessed on day 5 prior to, IPE, 3 h, and 6 h post eccentric exercise as well as the mornings of d 6-10. This assessment will be conducted while the subjects will resting following the blood draws. Subjects will ask to rate their overall quadriceps soreness on an ordinal 1-10 scale (1 = No Soreness, 10 = Unbearable Soreness; 6). In addition, quadriceps isokinetic exercise performance will be assessed at 24, 48, and 72 h post eccentric resistance exercise.
10- Tools and instruments:
Body Weight and Height
Each subject will be asked to wear shorts and remove shoes. Body weight will be recorded to the nearest 0.1 kg using a medical balance-type scale. Height will be recorded to the nearest 0.1 cm when the stadiometer portion of the scale is horizontal on the subject’s head while he’ll stand with his back to the scale.
Percent Body Fat
Body composition will be assessed using Harpenden skinfold calipers (Country Technology, Gays Mills, WI) and the protocol of Jackson and Pollack (1985). All measurements will take place on the right side of the body. The three sites viz., the chest, abdomen, and thigh will be used for measurement. The chest site will be a diagonal skinfold midway between the nipple and the anterior axillary fold. The abdominal site will a vertical skinfold located 2 cm to the right of the umbilicus. The thigh fold will be at a vertical skinfold midway between the proximal border of the patella and the inguinal crease. Each site will be measured to the nearest 0.1 mm, and the sites will rotate to prevent compression of subcutaneous fat. The average of 3 values within 1 mm will be averaged and a regression formula will be employed to determine percent body fat (Jackson and Pollack, 1985).
Peak oxygen uptake (VO2) will be measured using indirect calorimetry (CPX/D, Medical Graphics Corp., St. Paul, MN) and an incrementalprotocol on a cycle ergometer (Monarch 818E, Stockholm, Sweden). The initial resistance on the cycle flywheel will be with 0.5 kg. which will be increased by.5 kg following each 2 min stage. Heart rate and rhythm will be continuously monitored by telemetry, and heart rate will be recorded in every minute. Ratings of Perceive Exertion (RPE) (1) will be obtained at the end of every stage and at the time of test termination. The test will be terminated when the subject can no longer maintain the 60 rpm pedaling pace despite encouragement. Peak VO2 will be calculated as the average VO2 over the final minute of exercise.
Dominant leg isotonic one-repetition maximum (1-RM) will be measured on an isotonic leg extension station (Nautilus, Independence, VA). Each subject’s 1-RM will be determined by increasing or decreasing the resistance on the weight stack until the subject can only perform one repetition through full range of motion at a given weight. A 2.27 kg adapter will add to the weight stack to enhance precision. One minute of rest will be given between trials. Quadriceps peak torque, work performance with the maximal repetition, and total work for 5 maximal-effort repetitions will be measured on the dominant leg at a test speed of 60o/s using a Biodex isokinetic dynamometer (Biodex Corp., Shirley, NY).
24 h urine collections (sample) of the subjects for experimental days 3-9 will be made in polypropylene bottles with 1 ml 50% hydrochloric acid as a preservative. Collections will begin with the second void of the day and will be turned in each morning. Total volume will be recorded, and aliquots were frozen at -20oC. Urinary 3MH will be measured by an amino acid analyzer (PICO.TAG, Waters Association, Milford, MA). The interassay coefficient of variation (CV) will 3.1%. Urinary creatinine will be analyzed spectrophotometrically by manual assay procedure using a commercially available kit (Kit #555, Sigma Chemical Co., St. Louis, MO). All samples for a subject will be analyzed in duplicate during the same assay.
Blood samples (10 ml) will be drawn from the antecubital vein and will be placed into heparinized tubes. All samples will be placed in an ice bath for 30 min to clot and will be centrifuged at 3,000 rpm for 15 min at 4oC. Aliquots of serum will be stored at -20oC until analysis. All samples for a subject will be analyzed in duplicate during the same assay.
Serum glucose and serum CK will be analyzed spectrophotometrically using manual assay procedures from commercially available kits (Kits #520 and #47, respectively, Sigma Chemical Co., St. Louis, MO). Serum insulin, growth hormone (GH), testosterone, and cortisol will be analyzed by radioimmunoassay using commercially available kits (Diagnostic Products Corp., Los Angeles, CA). The intra-assay CV for GH will 17.9% while the interassay CV will 13.9%. All other intra-assay and interassay CV’s for the hormones less than 5%. Serum IL-1, IL-6, and TNF will be analyzed by high-sensitivity enzyme immunoassay using commercially available kits (Quantikine HS, R & D Systems, Minneapolis, MN).
The controlled diet will consist of a meat-free, 3-d rotating menu with a macronutrient content of 55 % carbohydrate, 30% fat, and 15% protein. A meat-free diet is indicated for measuring urinary 3-methylhistidine (3MH). Since 3MH is a an amino acid component of skeletal muscle, consumption of meat products can erroneously inflate 3MH values (Thomas, Ballard, and Pope, 1979;Young and Munro, 1978).
The menus will be developed by a departmental research associate who had prior experience in controlled feedings and will be based upon the protocols of Hickson et al.(1986). Dietary menus will construct using Nutritionist IV software (First Data Bank, San Bruno, CA). Each day’s menu will consist of a breakfast, lunch, and dinner at fixed times with snacks that could be eaten at flexible hours throughout the day. Meals will be prepared teaching classroom by the research associate or other trained foods personnel in the Department of Human, Nutrition, Foods, and Exercise. All food and beverage portions will be measured using measuring cups, measuring spoons, or an electronic balance scale. All food personnel will use gloves during preparation, serving, and cleanup. Since the subjects will not house overnight on, they will eat all of their breakfasts and most of their dinners in the teaching classroom and will be returned home each night. For any other meals not consumed in the classroom, the subjects will receive a small, insulated cooler packed with their pre-measured dinner, lunch, or snacks.
Initial energy of the diet will be set at 3,000 kcal/day. Subjects who experienced small weight losses and/or expressed hunger will supplement with Ensure liquid supplement (Abbott Laboratories, Columbus, OH) since it will be provided additional kcal while maintaining the macronutrient breakdown of the controlled diet. Subjects will instruct to consume no other foods, vitamin or mineral supplements, or alcoholic beverages during the experimental period. Diet record check-off sheets will turn in on a daily basis, and any Ensure consume or food not eaten will account for. All of these subjects’ diet records will be analyzed for caloric and macronutrient content using Nutritionist IV software.
On the day of the eccentric resistance exercise (day 5), diets will be adjusted in kcal and macronutient content to account for the experimental beverages so that the original caloric (3000 kcal/day) and macronutrient content (55 % carbohydrate, 30% fat, and 15% protein) will be maintained. Also, the timing of meals will be adjusted to accommodate the treatment beverages consumed that morning. A brunch meal will be the first solid food consumed 3 h post-exercise following a blood drain.
Data will be analized by using a repeated measures analysis of variance (ANOVA), followed by a Newman-keuls post hoc test in the presence of significant F ratio.
Figure 1. Study design.
All Experimental Days
Pre 1 2 3 4 5 6 7 8 9 10
D D D D D D D D D
W W W W W W W W W W W
U U U U U U U
B B B B B B B
MB MB MB
P P P P
S S S S S S
Experimental Day 5
Post-Resistance Exercise (h)
-0.5 IPE 1 2 3 4 5 6
B B B B
T T D D
S S S S
D - Controlled diet
U - 24-h urine collection
B - Blood draw
AE - Aerobic cycling: 40 min at 70% VO2peak, 5 1-min sprints
RE - 10 sets of 10 eccentric leg flexions (120% 1-RM)
P - Isokinetic performance test
MB - Muscle biopsy
W - Body weight
S - Muscle soreness assessment (1-10 scale)
T - Treatment (carbohydrate, carbohydrate-protein, or placebo
10. Time Scheduling
Preparing the proposal 1 month
Review of related literature 6 months
Preparing the tools 2 months
Data collection 5 months
Scoring 2 months
Analysis and interpretation 3 months
Report writing 3 months
Typing, Bending and submission 2 months
Total 24 months
Chapter 1: Introduction
Chapter 2: Review of related literature
Chapter 3: Methodology
Chapter 4: Analyses of data and interpretation
Chapter 5: Summary, conclusion and recommendation
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Research Student Co-Guide Research Guide
Ali Esmaeili khoshnezhad Dr.D.B.Tambe Dr.T.K. Bera
Research Project undertaking for Ph.D. in Physical Education in Pune University
Influence of Carbohydrate-branche chain amino acid on Glycogen Restoration, Hormonal response and Muscle Soreness Following By Acute Eccentric Exercise