NITROGEN BALANCE AND LEUCINE KINETIC APPROACHES TO THE EXAMINATION OF PROTEIN REQUIREMENTS AND PROTEIN METABOLISM IN RESISTANCE ATHLETES

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1 NITROGEN BALANCE AND LEUCINE KINETIC APPROACHES TO THE EXAMINATION OF PROTEIN REQUIREMENTS AND PROTEIN METABOLISM IN RESISTANCE ATHLETES By HARK A. TARNOPOLSKY, BPE., MD. A Thesis Submitted to the ~chool of Graduate studies in Partial FUlfilment of the Requirements for the Degree Doctor of Philosophy McMaster university May,

2 .' PROTEIN REQUIREMENTS AND METABOLISM IN RESISTANCE ATHLETES

3 DOCTOR OF PHZLOSOPHY (1991) (Medical Sciences, Cell Biology and Metabolism) McMASTER UNZVERSZTY Hamilton, ontario TITLE: Nitrogen balance and leucine kinetic approaches to the examination of protein requirements and protein metabolism in resistance athletes. AUTHOR: Mark A. Tarnopolsky, BPE. (McMaster University) MD. (McMaster University) SUPERVZSOR: Dr. S.A. Atkinson CO-SUPERVISOR: Dr. J.D. MacDougall NUMBER OF PAGES: xvi, 255.

4 ABSTRACT ;. Previous work has demonstrated that resistance exercise may increase dietary protein (PRO) requirements to a varying degree. Much of these discrepancies probably arose from differences in training intensity, classification of exercise, habituation to a training stimulus, and methodological considerations such as: 1. inadequate dietary adaptation periods; 2. failure to measure routes of nitrogen (N) loss (Le., sweat); 3. failure to measure the dietary PRO (nitrogen (N» conte~t; 4. collection periods too short to measure an impact of exercise (i.e., delay in N excretion due to dehydration); and 5. the use of measurements insensitive to short-term alterations in PRO metabolism (Le., lean body mass estimate from skinfolds to assess changes in muscle mass over a week of treatment). In addition, there is a paucity of information quantitating the influence of acute and chronic resistance exercise on PRO turnover. The overall purpose of this thesis was to examine N metabolism and the PRO requirements of resistance athletes, under well controlled conditions, with three discrete, but related studies. The first investigation was a randomized, cross-over study designed to determine the dietary PRO requirements of young men performing intensive body building resistance exercise during t~e early stages of training (novice) and to determine whether the consumption of excessive amounts of PRO had an iii

5 ergogenic (work enhancing) effect upon muscle mass/strength gains. Twelve inexperienced, young, male volunteers received either a PRO supplement (total PRO ln =2. 62 g kg-l d OI ) or a carbohydrate (CHO) supplement (total PRO IN =1. 35 g kgol d- I ) each for a period of 4 weeks during intensive (1.5 h d- I, 6 d-wk- I ) circuit weight training. Nitrogen balance (NBAL) measurements taken over the last 3 days on each diet were used to determine that the PRO intake (PRO IN ) for zero NBAL was 1.43 g-kgol d- I (+150=1. 62 g-kg-l d- 1 (recommended intake» _ Pre- and posttraining measurements of strength and estimates of muscle mass (density, creatinine excretion, and biceps muscle N content) were not different between diet treatments in spite of a significant training effect (increased strength and lean body mass) due to the exercise programme. It was recommended that young males performing body building-type resistance exercise require a dietary PRO IN of g.kg- 1 dol during the first 2 months of training and that there are no greater increases in 0- strength or muscle mass by consuming PRO in excess of this amount. The acute effects of resistance exercise upon leucine oxidation and whole body PRO synthesis (WBPS) were studied using stable isotope methodology in the second study. L-[l 13C]leucine was used as a tracer to calculate these variables in 6 healthy, fed, male athletes in response to a 1 h bout of circuit-set resistance exercise. The measurements were iv

6 performed prior to, during and for 2 h after exercise and corrections were made for the background 13C02/12C02 breath enrichment and bicarbonate retention factor (c). Results demonstrated significant increases in the background I3C02/12C02 breath enrichment at 1 and 2 h after exercise and in c during exercise. At 15 min after exercise c was significantly lower than at rest. There were no effects of exercise on leucine oxidation, WBPS, nor the rate of appearance of endogenous leucine or total leucine flux. We concluded that circuit-set resistance exercise did not affect the measured variables of leucine metabolism. In addition, large errors in calculating leucine oxidation and WBPS during resistance exercise can occur if background 13C02/12C02 breath enrichl!1ent and c are not accounted for. In the final study, leucine kinetics and NBAL were used to determine the dietary PRO requirements of sedentary (S) and resistance trained (BB) subjects. Each subject was randomly assigned to each of 3 dietary PROm (LP=O.86i MP=1.40i HP=2.40 g PRO kq-l d- 1 ) for a total period-of 13 days. Over the last J d, NBAL measurements were completed." and on the last day WBPS and leucine oxidation were determined from L-[1-13 C]leucine turnover. Regression analysis of the NBAL data was used to determine the PROm for zero NBAL for S=O. 69 g.kg"l dol and BB=1. 41 q.kg-i. dol and a recommended intake (ZERO intake +1 SO) for S=0.89 gokg-1 d- 1 and BB=1.76 q kg-i.d- l v

7 For BB the LP diet did not provide adequate PRO and resulted in an accommodated state (J. WBPS vs MP arid HP), the MP diet resulted in a state of adaptation as evident by the increase in WBPS (vs LP) and no increase in leucine oxidation (vs LP), while the HP diet did not result in increased WBPS compared to the MP diet but leucine oxidation did increase significantly indicating a nutrient overload. For S the LP diet provided adequate PRO and increasing PRO IN did not increase WBPS. Leucine oxidation increased for S on HP diet. Taken together, these results indicated that the MP and HP diets were nutrient overloads for S. There were no effects of dietary treatment on indices of lean body mass (creatinine excretion, body density) for either group. Overall, this research provided evidence that the PRO requirements of resistance athletes are greater than for sedentary individuals and are above current Canadian and u.s. recommended daily PRO IN for young, healthy.- males. The second study demonstrated that acute resistance exercise did not affect whole body leucine turnover and, in contrast to endurance exercise, i:here was no significant increase in leucine oxidation during exercise. In addition, this study demonstrated that changes occur in c and background breath enrichment during and after resistance exercise that can have an impact on leucine oxidation measurements. The final study also demonstrated that the positive NBAL that vi

8 occurs at high PRO~ does not appear to represent a physiological entity (i.e., no increase in WBPS or lean mass) but is probably an error in the NBAL method. vii

9 ACKNaiLEDGEHENTS.- I would like to thank my supervisor, Dr. S.A. Atkinson for her invaluable assistance in every aspect of my post-graduate training and I would lik~ to thank my co-supervisor, Dr. J.D. MacDougall for all of his input into the development of these studies and for his introducing me to the area of protein metabolism and exercise in I would like to thank the other members of my supervisory committee for their analysis of many aspects of this work: To Dr. H.P. Schwarcz for assistance in IRHS analysis and stimulating conversation on aspects of stable isotopes. To Dr. C.J. Toews for encouraging thoughtful analysis of my work. To Dr. C.J. Blimkie for kindly agreeing to act as supervisor during the last 6 months of the preparation of this thesis during Dr. MacDougall's sabbatical. Finally, I would like to thank those individuals who in many different ways contributed to the completion of this work: To my grandfather, Dr. J.G. Rempel, who instilled in me a love of science and nature and to my mother and father. To my daughters, stephanie and Alanna, whose childhood energy always gave me a reason to take a break from work and enjoy other aspects of life. To my friends, Alan chesley, stuart Phillips, Mike Waddinqton, Bruce Ainsworth, Gerrard Shoemaker and Cynthia cupido, without whom the completion of this thesis would likely not have been possible (and certainly not as enjoyable!). To the Canadian National Institute of Nutrition for financial support To Drs. R. Hill and J. Rosenfeld for analytical help. To the subjects who dedicated many hours to collections, infusions, and consumption of less than delicious meals. And to John sutton for continued encouragement. viii

10 .' TABLZ OF CONTENTS ABSTRACT ACKNaiLEDGEH.ENTS 4' " 'l'.'\ble OF CONTENTS.. LI:..1'l' OF FIGCJRBS 1". LIST OF TABL8s. LIST OF ABBREVIATIONS Chapter ~ a) b) c) d) e) GENERAL INTRODUCTION Introduction Historical perspective and rationale... Factors involved in determining the protein. requirements of athletes Potential mechanisms o~ increased protein requirements for endurance athletes Potential mechanisms of increased protein requirements for resistance athletes Duration and intensity of exercise..... The state of training of an individual. The energy and macronutrient content of. the diet Methods of determining protein requirements. objectives and hypotheses..... study 1 (Chapter 3) study 2 (Chapter 4). study 3 (Chapter 5) Chapter 2 - HODBLS AND IfB'l'BOD DBVBLOPIIENT stable isotope model... General isotope terminology Model of protein turnover.. Assumptions of the model Validity of the model.. stablg isotope methodology. Isoto~,ratio mass spectrometer (IRMSj. Breath analfisis with IRKS. Micromolar 1C0 2 enrichment determination. Gas chromatography/mass spectroscopy (GC/MS) and GC. Plasma a-kic derivatization and GC/MS acquisition parameters GC separation of muscle amino acids iii viii ix xii xiv xv ix

11 .- Chapter 3 - PROTEIN REQUIREHENTS AND LEAN BODY MASS DURING :INTENSIVE RESISTANCE TRAINING IN NOVICE BODY BUILDERS Introduction. Methods SUbjects Experimental protocol Lean body mass One repetition maximum Neuromuscular function Muscle biopsy Nitrogen balance. Biochemical analyses. statistical analyses Results. Dietary analyses. Nitrogen balance. Protein requirement. Anthropometry, creatinine excretion, and... biceps muscle nitrogen N~uromu~cular properties and strength data.. D~scuss~on Chapter 4 - WHOLE BODY LEUCINE HBTABOLISII DURING AND AFTER RESISTANCE EXERCISE IN FED IiUHANS a) b) Introduction. Methods SUbjects Experimental protocol.... Analyses Breath analysis Plasma analysis Calculations. statistical analyses... Results. Gas exchange Bicarbonate retention factor and. background breath enrichment Breath and plasma a-kic isotopic enrichments Calculations of leucine flux and oxidation; estimates of wbole body protein synthesis (~BPS) ~d proteolysis D~scuss~on x

12 Chapter 5 - A KINETIC AND NITROGEN BALANCE APPROACH TO THE DETERJfINATION OF PROTEIN REQUIREMENTS FOR RESISTANCE ATHLETES..' Introduction. Methods. SUbjects Experimental protocol Infusion protocol. strength measurements Lean body mass Nitrogen balance. Biochemical analyses Calculations. statistical analyses Results. Dietary analysis data Lean body mass, CPK activity, and strength Nitrogen balance.. Leucine turnover. 5.4 Discussion... Chapter 6 - OVERAI.L CONCLUSIONS REFERENCES APPENDICES I. II. III. IV V VI. Conclusions. Future research.... Tracer methodology NEAL and Diet Analyses Misc. A1~alyses Chapter 3 Data Chapter 4 Data Chapter 5 Data xi

13 LIST OF FIGURES Figure Description Page Figure 1 Protein turnover and muscle hype~trophy 19 Figure 2 Hypothetical response of protein turnover 36 to dietary intake.' Figure 3 Isotopic plateau in breath CO 2 and plasma a-kic enrichment Figure 4 Model of whole body leucine metabolism and 50 reciprocal pool model Figure 5 Bicarbonate retention factor and breath CO 2 background enrichment during exercise Figure 6 Effect of diet on breath CO 2 enrichment Figure 7 Figure 8 Figure 9 GC elution profiles and a-kic mass spectra o. 82 Theoretical a-kic derivative isotope cluster 0 83 abundance and mass calculation GC elution profile of authentic amino acid. 87 standards and human muscle hydrolysate Figure 10 Day to day urinary UN excretion Figure 11 Regression equation to determine PRO requirements Figure 12 Anthropometric, creatinine excretion, and biceps muscle N data Figure 13 Neuromuscular properties and strength data xii

14 LIST OF FIGURES (cont:.) Figure Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Description study design for Chapter Gas exchange data Bicarbonate retention factor and background breath CO 2 enrichment Breath CO 2 enrichment and plasma a-kic Leucine oxidation and WBPS Leucine flux and Ra endogenous leucine 143 Urinary UN excretion Regression equation to determine PRO 176 requirements WBPS and leucine oxidation 178 Total leucine flux and protein breakdown 179 Regression equation to determine PRO 198 requirements (all groups data).. xiii

15 LIST OF TABLES Table Table 1 Description SUbject characteristics (Chapter 3) 92 Table 2 Table 3 Dietary intake 101 Nitrogen balance 103 Table 4 Table 5 SUbject characteristics (Chapter 4) SUbject characteristics (Chapter 5) Table 6 Table 7 Table 8 Dietary intake 170 Lean body mass, CPK activity and strength Nitrogen balance xiv

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Examination of an Indicated Prevention Program. Targeting Emotional and Behavioural Functioning in. Young Adolescents

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