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Effect of energy restriction on substrate utilization: muscle function and myofibrillar protein degradation… Parkes, Sabina C. 1995

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E F F E C T OF E N E R G Y RESTRICTION O N SUBSTRATE UTILIZATION, M U S C L E FUNCTION A N D MYOFIBRILLAR PROTEIN DEGRADATION IN SEDENTARY M A L E S A N D ATHLETIC FEMALES  By Sabina C. Parkes B.Sc. Simon Fraser University, Burnaby, British Columbia, 1992  A THESIS SUBMITTED LN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES DIVISION OF H U M A N NUTRITION SCHOOL OF FAMILY A N D NUTRITIONAL SCIENCES  We accept this thesis as conforming to the standard  T H E UNIVERSITY OF BRITISH C O L U M B I A Spring 1995 © Sabina C. Parkes, 1995  In presenting  this  thesis , in  degree at the University of  partial  fulfilment  of  the  requirements  for  an advanced  British Columbia, I agree that the Library shall make it  freely available for reference and study. I further agree that permission for extensive copying of  this thesis for  department  or  by  his  or  scholarly purposes may be granted by the head of her  representatives.  It  is  understood  that  copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT  A n oxidative preference for fat or carbohydrate substrates has an impact on muscle protein metabolism. This study investigated how changes in substrate oxidation (through dieting) influences muscle function and contractile protein profiles. Eight sedentary males (Age:22 ± 1 years; Height: 175.9 ± 1.1 cm; Weight: 84.2 ± 2.9 kg) were placed on a diet and exercise program for 14 days where total energy deficit was 33% (22% from diet; 11% from exercise). All meals were consumed at the study center. Subjects jogged for 0.5 hours/day. Muscle biopsies and quadriceps muscle group function tests (Kin-Corn isokinetic dynamometer) were completed pre- and post-treatment Body weight decreased (1.5 ± 0.3 kg; p < 0.01) and fat oxidation increased (p < 0.05). Concentric average torque was lower (p < 0.05) at a movement velocity of 30 degrees/second. Myofibrillar proteins treated with a non-lysosomal protease, calpain, showed no increase in calpain activity within myofibrils. To test the hypotheses in an active population, this study was repeated in athletic females. Fourteen endurance athletes (Age:27 ± 2 years; Height: 167.2 ± 1 . 6 cm; Weight: 61.8 ± 1.9 kg; VC»2 max: 49.0 ± 0.9 ml/min/kg) were randomly assigned to maintenance energy (100% kcal) or energy restricted (75% kcal) diet groups. Subjects exercised for 100 minutes/day (within their normal exercise routine). Among the 75% diet group, body weight decreased (1.7 ± 0.3 kg; p < 0.05) and fat oxidation increased (p < 0.05). Peak concentric torque was significantly lower (p < 0.05) for the 75% diet group at 120 degrees/second, while average eccentric torque was significantly larger (p < 0.05) at 180 degrees/second. There were no significant changes in myofibrillar calpain activity in both diet groups. Results obtained from both groups suggest that increased fat oxidation induced by energy restriction was not conclusively related to changes in myofibrillar calpain activity. There were also no changes in quadriceps muscle function due to inconsistent trends within  both sets of data. Indirectly, elevated calpain activity may be related to decreased muscle function since lack of changes in calpain activity was associated with no changes in muscle function. However, a direct effect of calpain activity on muscle function remains to be demonstrated.  iii  TABLE OF  CONTENTS  ABSTRACT LIST  OF  ii TABLES  ix  LIST O F FIGURES  xi  ACKNOWLEDGMENT  xii  I.  INTRODUCTION  1. Rationale 2. Objectives 3. Hypotheses II.  III.  1 3 5  REVIEW OF LITERATURE  1. INTRODUCTION  6  2. DIET INDUCED CHANGES IN SUBSTRATE OXIDATION  7  3. EXERCISE INDUCED C H A N G E S IN S U B S T R A T E OXIDATION  10  4. S U B S T R A T E OXIDATION A N D PROTEIN D E G R A D A T I O N A) Related to an elevated energy expenditure B) Related to a low energy intake  17 19 25  5. PROTEOLYTIC SYSTEMS INVOLVED IN PROTEIN DEGRADATION A) Related to an elevated energy expenditure B) Related to a low energy intake  28 32 35  6  36  MUSCLE  FUNCTION  7. DIETING A N D ATHLETES  39  8. S U M M A R Y  41  EXPERIMENTAL DESIGN AND  1  S T U D Y DESIGN A) Males B) Females  METHODOLOGY  42 44 44  2. SUBJECTS A) Males B) Females  45 45 46  3. STUDY PROTOCOL  48  iv  METHODS 1. DIETARY INTAKE  49  2. ANTHROPOMETRICS A) Weight B) Height C) Body Mass Index D) Skinfolds i) Males ii) Females  50 50 51 51 52 52 54  3. EXERCISE INTERVENTION A) Exercise Protocol i) Males i) Females B) Energy Expenditure Determination i) Males ii) Females  56 56 56 57 57 57 58  4.  SUBSTRATE OXIDATION A) Resting Energy Expenditure B) Respiratory Quotient  ...58 60 60  5. M U S C L E FIBER ANALYSIS  61  6. M U S C L E  63  FUNCTION  STATISTICAL ANALYSIS OF T H E D A T A . .  68  SIGNIFICANCE  70  OF T H E STUDY  H U M A N STUDIES  IV.  70  RESULTS 1.  SUBJECTS! A) Males B) Females  71 71 73  2. DIETARY INTAKE A) Males B) Females  76 ....76 78  3. ANTHROPOMETRICS A) Males B) Females  81 81 83  4. EXERCISE D A T A A) M a l e s B) Females  86 86 91 v  5. S U B S T R A T E O X I D A T I O N A) Males B) Females  95 95 97  6. M U S C L E FIBER ANALYSIS A) Males B) Females  100 100 100  7. M U S C L E F U N C T I O N A) Males B) Females  101 101 104  8. F A C T O R S R E L A T I N G T O C H A N G E S LN C A L P A I N A C T I V I T Y A) Males B) Females  107 107 107  9. F A C T O R S R E L A T I N G T O C H A N G E S LN M U S C L E F U N C T I O N A) Males B) Females.  108 108 109  10.SUMMARY  118  DISCUSSION 1. INTRODUCTION  119  2. SUBJECTS A) Males i) Dietary Intake B) Females i) Dietary Intake  119 119 120 121 123  3. ANTHROPOMETRICS  125  4. EXERCISE A) Males B) Females  129 129 129  5. S U B S T R A T E  OXIDATION  130  6. M U S C L E P R O T E I N D E G R A D A T I O N  132  7. M U S C L E  137  FUNCTION  8. POSSIBLE RELATIONSHIPS ELUCIDATED F R O M THIS R E S E A R C H A) Substrate oxidation and calpain activity B) Muscle function and calpain activity vi  144 144 145  9. IMPLICATIONS  FOR F U T U R E R E S E A R C H  10. S T U D Y LIMITATIONS  148 149  CONCLUSION  150  REFERENCES  152  APPENDIX 1:  Menu schedule for male and female subjects  164  APPENDLX2:  Dietary analysis of the menu used in the study  165  APPENDIX 3:  Exercise recording form for female subjects  167  APPENDIX 4:  Advertisement used for recruiting male subjects  169  APPENDIX 5:  Advertisement used for recruiting female subjects  170  APPENDIX 6:  Consent form for male subjects  171  APPENDIX 7:  Consent form for female subjects  173  APPENDIX 8:  Dietary intake record instructions for female and male subjects  175  APPENDIX 9:  Food intake form  179  APPENDIX 10: Metropolitan height and weight tables, 1983  180  APPENDIX 11: Anthropometric measurement standards  181  APPENDIX 12: Equivalent percent fat for the sum of four skinfolds and  Canadian  norms  193  APPENDIX 13: Anthropometric recording form for male subjects  194  APPENDLX 14: Anthropometric recording form for female subjects  195  APPENDIX 15: Table of equation relating oxygen consumption and heart rate for the determination of energy expenditure for the male subjects  196  APPENDIX 16: Procedure for metabolic cart nutritional assessment  197  APPENDIX 17: Kin-Com data collection sheet  198  APPENDIX 18: Acceptance of ethics form  199  APPENDIX 19: Previous activities of male subjects  200  APPENDLX 20: Equation for calculation of ideal body weight  201  vii  APPENDIX 21: Run distance of female subjects during the study  202  APPENDIX 22: Activities of female subjects  203  APPENDIX 23: Time spent exercising by female subjects during the study  204  APPENDIX 24: Energy expended each during the study by each female subject  205  viii  LIST OF TABLES Page Table 1:  Table of reagents to add in determining myofibrillar calpain activity  62  Table 2:  Baseline characteristics of male subjects  72  Table 3:  Baseline characteristics of the female athletes according to the the dietary subgroup  74  Baseline anthropometric measurements of female athletes according to the dietary subgroup  75  Table 5:  Nutrient intakes of male subjects for four days  77  Table 6:  Mean nutrient intakes of the female athletes for four days, according to the dietary subgroup  79  Anthrompometric measures of male subjects before and after the dietary and exercise treatment  82  Anthropometric measures of female athletes according to the dietary subgroup before and after the respective diet period  84  Average energy expended per exercise session for male subjects  87  Table 4:  Table 7:  Table 8:  Table 9:  Table 10: Average exercise heart rate and reserve heart rate values for male subjects at various time points during the 30 minute exercise session  88  Table 11: Energy intake and energy expenditure of the male subjects  90  Table 12:  Activity characteristics of female athletes according to dietary subgroup during the study  91  Table 13: Average energy expended from exercise per day for female athletes according to the dietary subgroup  94  Table 14:  Resting energy expenditure data during diet treatment for male subjects  95  Table 15:  Post-prandial R Q values after consumption of a test breakfast for male subjects Resting energy expenditure data during diet treatment for female athletes according to the dietary subgroup  97  Table 17: Post-prandial RQ values after consumption of a test breakfast of female athletes according to the dietary subgroup  99  Table 16:  Table 18:  Myofibrillar calpain activities of female athletes  ix  96  according to the dietary subgroup before and after dietary period Table 19: Eccentric contractions of quadriceps muscles of male subjects before and after the diet and exercise period Table 20:  Table 21: Table 22: Table 23: Table 24: Table 25: Table 26: Table 27:  Table 28:  Table 29:  Table 30:  100 101  Concentric contractions of quadriceps muscles of male subjects before and after the diet and exercise period  102  Eccentric contractions of quadriceps muscles of female athletes according to the dietary subgroup before and after the diet period  104  Concentric contractions of quadriceps muscles of female athletes according to the dietary subgroup before and after the diet period  105  Post-diet substrate oxidation parameters correlated to the post-diet calpain activity in the 100% diet group  107  Post-diet muscle function parameters correlated to post-diet myofibrillar calpain activity in male subjects  108  Correlation coefficients for change in calpain activity to change in muscle function parameters for the male subjects  109  Pre-diet muscle function variables correlated to pre-diet myofibrillar calpain activity in the entire group of athletic females  110  Pre-diet muscle function variables correlated to pre-diet myofibrillar calpain activity in athletic females in the 100% diet group  112  Post-diet muscle function variables correlated to post-diet myofibrillar calpain activity for athletic females in the 100% diet group  114  Post-diet muscle function variables correlated to post-diet myofibrillar calpain activity for athletic females in the 75% diet group  116  Correlation coefficients for change in calpain activity to change in muscle function variables for the female athletes in the 75% diet group  117  x  LIST O F FIGURES Figure 1:  Study  Protocol  34  Figure 2:  Energy expenditure from exercise throughout the diet period  xi  93  ACKNOWLEDGEMENT  I would like to sincerely thank my supervisor Dr. Linda McCargar for her encouragement, guidance, and support, without which, this thesis could not have been completed. She has been an exceptional 'mentor', role model, and a good friend. I will never forget the many experiences that were shared: I will value them always. Additionally, I would like to express my most grateful appreciation to Dr. Angelo Belcastro who has treated as 'one of his own'. His knowledge, patience, and belief in me was essential to the production of this thesis. Furthermore, I would like to thank the students in the Belcastro lab whose undying support brought me through many rainy days. I would also like to thank Dr. Susan Barr for the invaluable input and continual interest that she offered as a committee member. I gratefully acknowledge the Canadian Fitness and Lifestyle Research Institute for funding this project. Finally, an extra special thanks goes to my parents (Corinna and Dave Goodman), my grandparents (Mary Anne and Bob Cooper), Amei Parkes, Michael Goodman, Kristin Smith, Sage Kennedy, Carolyn Sluis-Meyer, and Sylvia Gubeli whose unconditional support gave me strength I didn't realize I'd had. I would also like to express my gratitude towards my fellow graduate students, with special mention to Charatini Orphanidou, Elli Mackay, and Jamie Staples, who were always supportive and willing to lend a helping hand. I would also like to thank Lieu, Maya, and Meaghan, whose expertise contributed to the completion of the thesis. Finally, I would like to express my deepest gratitude to the subjects of the study, who were wonderful!  xii  INTRODUCTION  Chapter I INTRODUCTION: 1.  RATIONALE In 1988 the Canada's Health Promotion Survey technical report by H. Neilson  stated that more than 50% of Canadians want to lose weight. Because there is an increasing amount of concern about weight control among Canadians, Neilson concluded that Canadians participating in weight control programs need information regarding the degree of risk and types of problems associated with a combined weight reduction and fitness program. This has implications for the performance capability of individuals involved in a weight loss program who engage in physical activity (through work and an exercise program). The projects outlined in this study were designed to test whether a combined energy deficit (restricted diet and exercise) selectively alters the protein composition of muscle and whether this results in a decrease in overall muscle function. This information may have significant physiological impact on diet therapy and/or intervention programs for individuals who wish to lose weight but must continue to perform muscular work at prescribed work rates. Weight is an especially important concern in the female athlete population in that a lower body weight has often been equated with "better" performance. For example, there is an increased demand to be thin when: an athlete is judged on the basis of how she looked during a performance, or an athlete is involved in endurance sports and a lower body weight is equated with a faster time for a given distance (Wilmore, 1991). The fact that a low calorie intake may be a problem in the athletic population is demonstrated by the fact that as many as 25 % of athletes have an eating disorder (Wilmore, 1991). Knowledge regarding the effect of a low calorie intake in an athletic population may provide useful  1  INTRODUCTION information for athletes, coaches and parents regarding healthy dietary/exercise regimens that will optimize muscle performance. Thus, the main objective of this study was to assess whether changes in fat oxidation (induced by an energy restriction plus elevated energy expenditure) causes selective breakdown of myofibrillar proteins. Previous studies (Belcastro et al, 1988; Parkes et al, 1993) have shown that myofibrillar protein degradation is elevated when fat oxidation is increased as a result of an energy restriction in the form of diet. These changes in muscle proteolysis may there have an effect on muscle function. In summary, Chapter LT will provide background information on changes in fat oxidation with diet and exercise, myofibrillar proteolysis, and muscle function. Chapter U l discusses the methods used in the study. Chapter IV describes the results of the study, while chapter 5 discusses the results.  2  INTRODUCTION 2. PURPOSE OF THE STUDY  OBJECTIVES  (MALES)  1) To assess rates of fat oxidation with a 33% energy deficit (i.e. moderate diet restriction plus moderate aerobic exercise).  2) To measure alterations in muscle proteolysis by evaluating calcium stimulated protease activity (calpain-like activity).  3) To measure muscle function by assessing quadricep/hamstring capabilities with a maximal knee extension and flexion as well as an endurance test using a Kin-com apparatus.  3  INTRODUCTION OBJECTIVES (FEMALES)  1) To assess rates of fat oxidation in female endurance athletes who are/are not receiving 25% dietary energy restriction.  2) To measure alterations in muscle calcium stimulated proteolysis by evaluating calpainlike activity in female endurance athletes who are/are not receiving a 25% dietary energy restriction.  3) To measure quadriceps muscle group function by assessing quadricep capabilities performing a knee extension and flexion at maximal effort, and at submaximal effort as an endurance test, using the Kin-com apparatus in female endurance athletes who are/are not receiving a 25% dietary energy restriction.  4  INTRODUCTION 3.  HYPOTHESES  The null hypotheses for this study were:  Hypothesis 1: Changes in fat oxidation, induced by dietary restriction and aerobic exercise, result in no difference in proteolysis of skeletal muscle.  Hypothesis 2: Changes in fat oxidation, induced by dietary restriction and aerobic exercise, result in no difference in quadriceps muscle group function.  5  REVIEW OF LITERATURE  Chapter II  REVIEW OF LITERATURE  /.  INTRODUCTION The maintenance of sufficient ATP levels, which is essential for survival and  function of mammalian cells, is coupled tightly to the rates of substrate oxidation (Flatt, 1977). Because the rates of substrate oxidation are determined by the availability of substrate, the amount of energy provided should direcdy influence the viability and function of mammalian tissue. In the past, these issues in human nutrition and performance led to research which concentrated on energy balance, where maintenance of physiological function, tissue growth, and survival were believed to be adequate provided that enough energy was available. The nitrogen balance method was also used as a research tool in this area when it was identified that amino acids are essential for directed tissue growth and maintenance. Nitrogen and energy balance methods have gained support in nutrition studies from a practical perspective. However, from a theoretical perspective, they offer limited information regarding the mechanism (s) underlying the role of energy restricted diets on substrate oxidation or on the potential metabolic repercussions for specific tissue/organ function. It has been reported that energy restriction may cause changes in substrate oxidation, which may subsequently alter muscle composition and function (Butterfield, 1987). A selective degradation of functional muscle proteins may occur as a result of the reduced energy state (Dahlmann et al, 1986; Frayn and Maycock, 1979). This may have  6  REVIEW OF LITERATURE implications for people who are energy restricted and are required to maintain specific physical performance levels. Athletes are one such group of people where specific performance levels may be compromised by a low energy intake. The combined effects of high energy expenditure and low energy intake may increase fat utilization, which in turn may have compounding effects on muscle protein degradation and therefore, muscle performance. Whether the effect of an increase in fat oxidation induced by an energy deficit (in the form of an energy restriction or energy restriction plus increased energy expenditure) causes selective degradation of identifiable functional proteins in muscle has not been examined in humans. This is the primary focus of this study. The literature review examines this question. Parts two and three of the literature review deal with changes in substrate oxidation as a result of an energy restriction in the form of diet and exercise, respectively. The impact that this increase in fat oxidation has on myofibrillar protein degradation is discussed in Part 4. The possible mechanism (s) responsible for the increase in muscle proteolysis as a result of an energy restriction is discussed in section 5. Part 6 describes the effect of an energy restriction and an increase in calpain activity on muscle function. Finally, Part 7 examines the prevalence of a low energy intake in the athletic population.  2.  DIET INDUCED CHANGES IN SUBSTRATE OXIDATION The purpose of this section is to assess changes in substrate oxidation induced by  changes in diet. The quantity and source of macronutrients influences the type of substrate utilized and the rates of substrate oxidation (McCargar et al, 1989). For example, protein ingestion activates both protein degradation and protein synthesis. In fact, protein and non-protein energy may stimulate protein synthesis and/or degradation through different mechanisms  7  REVIEW  OF  LITERATURE  and therefore may have an additive effect on protein turnover (McCargar et al, 1989). Thus, non protein energy, while affecting nitrogen retention, affects protein turnover in a different manner than protein energy (Reeds and Fuller, 1983). Ingestion of non-protein energy in the diet influences nitrogen balance. For example, Richardson et al (1979) reported that when carbohydrate, as opposed to fat, was the major energy source in a liquid diet (daily energy intake was less than 50 kcal/kg) fed to healthy male subjects, the most efficient nitrogen utilization was achieved. At higher energy intakes (above or equal to 50 kcal/kg body weight, for example), the opposite was observed (fat, as the major energy source, achieved the most efficient nitrogen utilization). McCargar et al, (1989), found that when increasing the fat content in the diet (and thus decreasing the CHO:fat ratio) there was an increase in nitrogen retention and fat oxidation and a slight decrease in energy expenditure. Acheson et al (1984) also reported a blunted thermogenic effect of a meal following exposure to a high fat (75% of total energy) diet. The thermic effect of feeding is one method used to quantify rates of substrate oxidation. It is the energy expended in excess of resting metabolic rate for the digestion, absorption, transport, metabolism, and storage of food and it accounts for approximately 10% of daily energy expenditure (Van Zant, 1992). Findings of McCargar et al (1989) were supported by findings of Phinney et al (1983) who showed that there was a significant increase in fat oxidation after feeding healthy lean subjects a high fat (84% of total energy) diet at maintenance energy levels for 4 weeks. This preferential shift to fat oxidation for energy spares nitrogen losses as the reliance on glucose and/or amino acid oxidation for energy is decreased. McCargar et al (1989) also found that when subjects were fed a high carbohydrate diet, there was an increase in oxygen consumption and carbon dioxide production as compared to subjects ingesting a high fat diet. These findings in combination with the facts that the thermogenic response to feeding and lipogenesis are both increased with a high  8  REVIEW OF  LITERATURE  carbohydrate diet (CHO), contribute to the elevated energy expenditure seen with a high carbohydrate diet. This increase in metabolism associated with a high carbohydrate diet promotes an increase in amino acid oxidation and subsequent nitrogen excretion. The amount of energy consumed also affects the rate of substrates oxidized. For example, both starvation (Mole', 1989) and semistarvation (Mole', 1990) have been shown to decrease the resting metabolic rate (RMR: the energy necessary to maintain the physiological systems at rest (Van Zant, 1992) between 15 to 30%. This decrease in resting metabolic rate with energy restriction is not immediate. Stock (1986) did not find a change in RMR after 32-36 hours of fasting. Bray (1969) found that restricting energy intake from 3500 kcal/day down to 1900 kcal/day decreased RMR by 15% after 3 weeks of dieting, but RMR was unchanged during the first 4 days of restriction. Similarly, Mole' et al (1989) showed that RMR did not change relative to a 10day control period until day 5 of a calorie-restricted diet (500 kcal/day). Thus, unlike overfeeding which enhances RMR after 24 hours, underfeeding may require several days before R M R is reduced significantly (Mole', 1990). A low energy intake also affects the types of substrates which are oxidized. For example, Nelson et al (1992) found that a larger percent of resting energy expenditure was derived from lipid after a 5 month diet restriction in obese females. Furthermore, Froidevaux et al (1993) found that the resting RQ value decreased in moderately obese women as a result of an energy restriction involving diet and exercise. Thus, the results obtained from these studies show that an energy restriction promotes the utilization of fats as an energy source under resting conditions. A low energy intake may also affect what fuels are metabolized after the ingestion of food. For example, Froidevaux et al (1993) found that the post-absorptive R Q decreased in 10 moderately obese women who lost weight due to a restrictive energy intake. They also found that the rate of lipid oxidation measured over a 24 hour time period, increased significantly during dieting.  9  REVIEW OF  LITERATURE  Although it is well established that an energy restriction in obese individuals results in an increased fat utilization under resting conditions and after the ingestion of food, the effect of an energy restriction on the types of substrates utilized under resting conditions and after the ingestion of food in endurance trained athletes and normal weight individuals remains to be determined.  3.  EXERCISE  INDUCED  CHANGES IN SUBSTRATE  OXIDATION  This section examines the effect of prolonged low intensity exercise and a trained state on the substrates utilized during exercise. Glucose (stored as glycogen), and fatty acids (stored as triglycerides) are the only quantitatively important energy sources during exercise. Of these, glycogen is the more important fuel for strenuous exercise that requires 65% to 100% V O 2 max (Vranic and Lickley, 1990). Thus, as exercise intensity increases, a shift in substrate utilization towards carbohydrate occurs, even in the trained state (Brooks and Mercier, 1994). Liver glycogen is important because it is the major source of blood glucose, which is the primary substrate for the central nervous system (Vranic and Lickley, 1990). Endurance exercise training increases the capacity to use all classes of energy substrates (Brooks and Mercier, 1994). For example, it has been suggested that athletes are better able to regulate the balance between glucose mobilization and utilization via the hormonally induced extraction of glucose from liver glycogen (Vranic and Lickley, 1990). Near the end of prolonged exercise, athletes do not show a continuous fall in arterial glucose as seen in untrained persons, but rather a slight rise has been observed (Keul et al, 1972). It is also possible that the increased free fatty acid (FFA) levels (Vranic and Lickley, 1990), produced as a result of triglyceride lipolysis during exercise, may inhibit the extraction of glucose from liver glycogen and its subsequent oxidation; this constitutes the glucose-fatty acid cycle (Brooks and Mercier, 1994).  10  REVIEW OF LITERATURE Glycogen content is also thought to be increased after a training program (Vranic and Lickley, 1990). Short et al (1969) found that after 6 weeks of training there was a 40% increase in muscle glycogen in the working leg. Thus, due to the increased substrate utilization occurring during athletic training, a higher storage capacity of the endogenous substrate, glycogen, may occur in the muscle cell (Vranic and Lickley, 1990). During prolonged strenuous exercise, for which muscle glycogen utilization is essential, fatty acids serve as an important secondary fuel. This results in a sparing of glycogen and prolongs the exercise time before glycogen depletion and exhaustion occur (Vranic and Lickley, 1990). Overall, the proportion of fat contributing to oxidative metabolism during prolonged exercise has been reported to be between 40 and 80% (Young et al, 1967). During rest and mild- to moderate-intensity exercise, lipids predominate as energy sources, especially in the endurance trained state. The supply of free fatty acids to the working muscle is promoted by physical training (Cobb and Johnson, 1963). Endurance exercise training increases the subject's capacity to oxidize lipids during exercise. For example, Keul et al (1963) found that trained animals had a significantly increased oxidation of palmitic acid (Keul et al, 1972). It was also found that the plasma F F A concentration was generally higher in the trained, compared to the untrained state during submaximal exercise at the same absolute intensity (Costill et al, 1973; Winder et al, 1979). Thus, during prolonged exercise, trained muscle utilizes FFAs for energy production to a greater extent than untrained muscle. This is reflected in a lower respiratory quotient (Hermansen et al, 1967). It was found that an increased capacity of trained muscle to oxidize blood borne FFAs and triglyceride along with an increased capacity to access intramuscular triglycerides results in glycogen sparing (Hermansen et al, 1967) and increased exercise endurance (Brooks and Mercier, 1994; Randle and Garland, 1963) in humans (Hermansen et al, 1967; Rennie et al, 1976) and rats (Randle and Garland, 1963; Randle et al, 1964). This  11  REVIEW OF  LITERATURE  increase in endurance in trained subjects appears to be a result of the increased mitochondrial reticulum mass (Brooks and Mercier, 1994). Specifically, training increases the enzymes required for: F F A translocation, the T C A cycle, the beta-oxidation path (Brooks and Mercier, 1994). Training also increases the components of the electron transport chain needed to oxidize FFAs (Brooks and Mercier, 1994). There is also a simultaneous inhibition of glucose uptake, glycolysis, glycogenolysis, and pyruvate oxidation (Randle and Garland, 1963; Randle et al, 1964) as a result of down regulation (through substrate inhibition; Brooks and Mercier, 1994) of cytoplasmic phosphofructokinase and pyruvate dehydrogenase (Havel et al, 1967; Phinney et al, 1984). Thus, use of anaerobic energy systems such as anaerobic glycolysis and phosphocreatine in providing energy for endurance exercise is not significant or facilitated (Fox, 1984). The source of additional triglycerides utilized in the trained state is thought to originate from intramuscular triglyceride stores as it has been proposed that the trained muscle has an increased capacity to access intramuscular triglycerides (Brooks and Mercier, 1994). Furthermore, additional utilization of triglycerides in trained muscle may be due to metabolic adaptations in adipose tissue. Savard et al (1986) did biopsies of supra-iliac adipose tissue in male marathon runners and sedentary controls and found that the runners had greater lipoprotein lipase activity in the adipose tissue along with greater basal and insuhn-stimulation of conversion of glucose to triglycerides. The time course of these adaptive changes in substrate utilization as a result of becoming trained has not been defined. For example, it is unknown whether described enzymatic changes in lipid and carbohydrate metabolism may occur after 1 week of training in previously sedentary males. Therefore, the changes in fat oxidation occurring as an individual becomes more trained needs to be established. It is unknown as to whether the increased utilization of fats in the trained state offers any physiological advantage. It may be that it is only a compensatory mechanism for the decreased beta-adrenergic stimulation of lipolysis in adipose tissue (Vranic and Lickley,  12  REVIEW OF LITERATURE 1990). It has also been suggested that a reduced sympathetic nerve stimulation (resulting in a decreased beta-adrenergic response) after training results in a decrease in glycogenolysis and attendant decreases in carbohydrate utilization during exercise (Brooks and Mercier, 1994). This in turn would promote the utilization of FFAs and triglycerides as fuels for the working muscle (Brooks and Mercier, 1994). The amount of F F A utilized during exercise is determined by the energy requirement and the level of substrate (Vranic and Lickley, 1990). For example, when glycogen stores are low, the substrate supply is no longer sufficient to cover energy cost, the triglycerides are hydrolyzed to fulfil that purpose. When there is a lack of energy substrates in the nutritive medium, triglycerides are broken down and utilized. Havel et al (1967) found that in fasted subjects, fat accounted for 40-50% of total energy supply (low glucose levels occurring during prolonged exercise may promote the increase in FFAs in the plasma). Thus, fatty acids can also function as a primary fuel (Havel et al, 1967). This makes it possible for exercise to be continued for longer duration, even in the fasting state (Costill et al, 1973). When subjects were placed on a high carbohydrate diet, fat accounted for only 10% of total energy supply (Cobb and Johnson, 1963). Shifts in the choice of fuel oxidized during exercise can be modified by the type of food consumed (Gollnick and Bayly, 1986; Wagenmakers et al, 1991). A shift towards a greater use of fat during exercise can be induced simply by reducing the concentration of glycogen stored in muscle (Gollnick et al, 1981). In these experiments the low glycogen concentrations were produced in one leg by a previous day's one-legged exercise and the consumption of a low carbohydrate diet. During two-legged exercise on the second day, the respiratory quotient, obtained through indirect calorimetry,was lower in the subjects whose leg had low glycogen as compared to the subjects whose leg had normal glycogen levels. The uptake of FFAs was also greater in the glycogen depleted leg. Therefore, it was suggested that chronic consumption of a low carbohydrate diet can produce alterations  13  REVIEW OF  LITERATURE  in the concentrations of key enzymes in muscle that would promote the use of fat during exercise (Gollnick and Bayly, 1986). Overall, substrate utilization patterns during exercise of graded intensities are determined by a number of factors. These include the following (Brooks and Mercier, 1994): sympathetic nervous system initiated events along with those initiated by actions of insulin and glucagon; blood lactate level; dietary and exercise histories; size of glycogen depots; cardiac output (determined by exercise intensity and duration); blood flow distribution; muscle recruitment; muscle mitochondrial mass. The exercise bout itself, and the training status of the individual may affect the rate at which substrates are utilized. For instance, it is well known that oxygen consumption and energy expenditure remain elevated above resting values for a period of time after a single bout of exercise. The magnitude and duration of the elevation of postexercise energy expenditure has been the focus of considerable research, although the findings are inconsistent. Several studies (Brehm and Gutin, 1986; Freedman-Akabas et al, 1985; Pacy et al, 1985) suggest that metabolic rate returns to pre-exercise/resting conditions within the first hour after exercise, whereas other studies have indicated that metabolic rate may remain elevated for a much longer period of time after exercise (Bahr et al, 1987; Bielinski et al,1985; Maehlum et al, 1986; Tremblay et al, 1988). For example, Poehlman et al, (1989) examined the effect of acute exercise on metabolic rate. Six untrained men performed a 90-minute bout of exercise on a cycle ergometer at 50% of VIX, max. Metabolic rate was measured at rest (control period), and 24, and 48 hours after exercise. There was no difference in RMR 24 and 48 hours after exercise compared to the control condition implying that low intensity acute exercise of 90 minutes duration has no sustained effect on RMR in the late postexercise recovery period. In contrast, Maehlum et al (1986) demonstrated that metabolic rate in young men and women was significantly higher during the 12 hours following a 65-90 minute bout of bicycle exercise at 70% V 0  9  max compared to the 12 hours following a 90 minute supine  14  REVIEW OF LITERATURE rest. Metabolic rate was also higher 24 hours after the exercise bout compared to 24 hours after the controlled resting condition. Wolfe et al (1990) described the contribution of the energy cost of the triglyceridefatty acid cycle to energy expenditure during and after exercise. This substrate cycle consists of cleaving the triglyceride molecule into the glycerol and fatty acids in the adipocyte, followed by re-esterification of fatty acids to triglyceride which may occur in the adipocyte (intracellular) or the liver (extracellular). Five healthy males were studied during 4 hours of walking at 40% VC«2 max and for 2 hours postexercise. At the end of the 2 hour recovery period energy expenditure remained elevated above pre-exercise level by 20%. The energy cost of the triglyceride cycling was 0.8 kcal/h at rest, 1.7 kcal/h during exercise, and 4.7 kcal/h during recovery. The energy cost of the triglyceride-fatty acid cycle therefore became significant during the recovery period, contributing 13.7% of the postexercise metabolic rate. This study suggests an important role of the triglyceride-fatty acid cycle in postexercise energy metabolism in healthy subjects. It can only be speculated that the same response would occur in trained individuals. The amount of time in which fat utilization remains elevated after an exercise bout has not been characterized in athletes or previously sedentary individuals. Additionally, the type of substrates utilized in trained individuals and untrained individuals (put on an exercise program) during daily activities and after consumption of food remains to be established. The subjects' fitness level may affect the change in the RMR measured postexercise. Several studies have examined differences in RMR between trained and untrained individuals and have obtained conflicting results. LeBlanc et al (1984) measured resting oxygen consumption in 30 females separated into untrained (n=10), moderately trained (n=10) and highly trained females (n=10). All women were tested during the postovulatory period of their menstrual cycle and resting oxygen consumption was measured using indirect calorimetry. Although the authors reported no differences between  15  REVIEW OF LITERATURE the groups, there was a trend for resting oxygen consumption (adjusted for body weight) to increase with the level of training. Poehlman et al (1988) found that RMR, expressed per kilogram of fat-free weight, was higher in aerobically trained men compared to untrained men. Using a ventilated hood system the R M R of 9 trained and 9 untrained weight stable men was compared 24 hours after the last exercise bout Resting metabolic rate adjusted per kilogram of fat-free weight was 11 % higher in trained men than untrained men. Although the mechanism for the higher RMR in highly trained people remains to be established, Poehlman et al (1991) hypothesized that RMR may be affected due to the high caloric turnover concurrent with the maintenance of energy balance. This 'high energy flux state' would be accomplished by matching a high level of food intake with a high level of exercise-induced energy expenditure, which is characteristic of weight stable endurancetrained athletes (Poehlman et al, 1991). The cross-sectional studies do not provide a clear picture regarding the effects of the 'trained state' on RMR due to many confounding variables. The factors which contribute to discrepant findings may include (Poehlman et al, 1991): insufficient sample size; the timing of indirect calorimetry measurements relative to the last exercise bout; technical or methodological errors in determining energy expenditure; or within subject variability due to preceding dietary practices. The effect of a diet and exercise program on the rate at which energy is utilized yields conflicting results. Belko et al (1987) found that the RMR was not altered by an exercise and energy restriction program compared to an energy restriction only regimen in moderately overweight women confined to a metabolic unit. In both groups, energy intake and expenditure were manipulated to result in an energy deficit of 50% for 6 weeks. The authors concluded that despite a 50% energy deficit achieved by diet alone or diet in combination with exercise, no energy conservation mechanisms were evident.  16  REVIEW OF LITERATURE Nieman et al (1988) however found that 21 mildly obese women during a 5 week exercise program and a 1300 kcal/day diet showed a 6% increase in R M R (kcal/day) compared to a control group who were consuming the same diet with no exercise. These results suggest that RMR may be enhanced when a caloric restriction resulting in moderate weight loss is combined with exercise training of sufficient intensity. The effect of a combined diet and exercise program in a trained and untrained normal weight population on RMR has not been characterized. In summary, it has been shown that prolonged low intensity exercise and a trained state promote the utilization of fat as an energy source during exercise. However, the effect of this on the type of substrates utilized after the ingestion of a meal requires further investigation. Furthermore, the combined effect of a diet and exercise program in trained and untrained individuals on substrates utilized after consumption of food also needs to be established.  4.  SUBSTRATE OXIDATION AND PROTEIN DEGRADATION The increases in fat oxidation induced by an energy restriction may be associated  with increases in muscle proteolysis. A concrete relationship between these two variables has not been established. This section investigates the impact of an increase in fat oxidation, induced by an energy restriction (in the form of a low energy intake, elevated energy expenditure, or a combination of both), on muscle proteolysis. The equilibrium of the rate of protein synthesis and protein degradation determines protein metabolism. Any one- sided change in these rates will favor either synthesis or degradation. For example, when animals and humans are subjected to adverse dietary conditions such as food deprivation, the result is a negative nitrogen balance (Young et al, 1967) originating from striated muscle. In this situation the energy requirement is not being met, nitrogen is lost from the body, and the protein lost from the body is more than  17  REVIEW OF LITERATURE the protein intake. This implies that greater than normal amounts of protein degradation are occurring in muscle under the condition of a low energy intake. Pathological states associated with altered metabolic status have been found to cause a one-sided change in reaction rates. For example, increases in fat oxidation induced by diabetes may result in increases in the activity of calpain (calcium activated neutral protease: a non-lysosomal protease capable of degradating myofibrillar proteins) (Kettelhut et al, 1994) and calpain degradation rates in the Z-line area of a myofibril. It was suggested that the 'more oxidized' (lower sulfhydryl group reactivity and content) diabetic cardiac myofibrils produced a more susceptible substrate for calpain (Belcastro et al, 1994). Thus, the altered oxidation-reduction status induced by diabetes has been suggested to be a targeting mechanism for selected protein degradation (Belcastro et al, 1994). There is also indirect evidence that the Z-line region of the muscle is selectively affected when fat oxidation is increased as a result of fasting (Dahlman and Reinauer, 1981) and exercise (Belcastro, 1993). Although hormone activity has been shown to directly control protein synthetic and degradative processes and therefore total amount of protein in muscle (Vranic and Lickley, 1990), it is becoming increasingly apparent that the oxidation of fat or carbohydrate substrates occurring, as a result of diet manipulation, impacts protein metabolism independent of the hormonal changes (Goldberg et al, 1980; Short et al, 1969). The mechanism (s) underlying the changes in protein metabolism occurring as a result of changes in fat and/or carbohydrate metabolism are not known. Lowell and Goodman (1987) investigated this question by inhibiting lipid metabolism using nicotinic acid (which is an antilipolytic agent). Nicotinic acid administration resulted in increased protein degradation and decreased protein synthesis in muscle. Because protein degradation was elevated when lipid metabolism was inhibited, they concluded that under uninhibited conditions lipid fuels directly modulate protein metabolism in muscle by attenuating the breakdown of myofibrillar protein independent of their oxidation as a fuel source for  18  REVIEW  OF  LITERATURE  muscle. However, nicotinic acid administration was also accompanied by a decrease in plasma insulin levels (Garza et al, 1976) and an increase in plasma corticosteroid levels (Lowell and Goodman, 1987). Thus, a role of insulin and/or glucocorticoids in causing the protein changes seen with nicotinic acid treatment could not be ruled out. It was postulated that free fatty acids may specifically modulate the myofibrillar protein degradation in two ways (Lowell and Goodman, 1987). Free fatty acids may directly attenuate the breakdown of protein in skeletal muscle. Free fatty acids may regulate the levels of insulin and/or glucocorticoids in plasma, resulting in an indirect regulation of the breakdown of proteins in skeletal muscle. It is also possible that the change in hormone levels themselves cause the changes in FFA levels, which may then regulate protein breakdown in skeletal muscle in some way. Although it cannot be shown that lipid fuels directly attenuate the degradation of protein in skeletal muscle independent of hormonal changes, Lowell and Goodman (1987) strongly suggest a direct role. A) Elevated Energy Expenditure The increased utilization of fat promoted by prolonged endurance training and a trained state may result in increases in myofibrillar protein degradation during and/or after exercise. This section examines the effect of exercise training (associated with an increased fat oxidation) on myofibrillar protein degradation. It is difficult to state whether muscle proteins, under well defined conditions, can be used as a fuel or rather as a precursor of muscle fuel during exercise (Keul et al, 1972). The energy demands of skeletal muscle during exercise can be almost entirely met by the degradation of glucose, glycogen and FFA. The extent to which proteins are used is dependent on duration and intensity of exercise, levels of glycogen stores, and the levels of energy intake (Randle et al, 1963). One way in which muscle protein degradation can be assessed is by looking at the branched chain keto acid dehydrogenase (BCKAD) activity in muscle. Branched chain keto-acid dehydrogenase is the rate limiting enzyme for the oxidative decarboxylation of  19  REVIEW OF LITERATURE branched chain amino acids (BCAA) in muscle (Randle et al, 1964). Branched chain keto acid dehydrogenase is found on the inner mitochondrial membrane and is regulated by phosphorylation and dephosphorylation, where the active form is the dephosphorylated form (Kasperek and Snider, 1987). It catalyzes the irreversible oxidative decarboxylation of branched chain keto-acids like alpha-ketoisocaproate, alpha-keto-beta-methylvalerate, and alpha-ketoisovalerate - all formed from B C A A transamination (Kasperek and Snider, 1987). Branched chain amino acids comprise about 35% of the indispensable amino acids in muscle proteins (Shimomura et al, 1990). Energy deficits can result in significant increases in rates of muscle protein degradation during or after exercise. Kasperek and Snider (1987), investigated the effects of an energy deficit and found that there was a 482% increase in B C K A D activity in the muscle of starved rats who exercised and only a 142% increase in enzyme activity in fed rats who exercised compared to rested rats. It was found that starvation itself does not increase B C K A D activity - but enhances the exercise induced activation. The activation of B C K A D in muscle during exercise means that the transamination of branched chain amino acids is elevated. This may occur as a result of the increased breakdown of myofibrillar proteins to branched chain amino acids. Thus, the amino acids produced by protein degradation could supply a significant fraction of the increased energy expenditure during exercise. The effects of low energy intake on increased muscle protein degradation may occur due to glycogen depletion which is often associated with low energy intakes. When human subjects cycled for 1 hour at 61 % V O 2 max in a glycogen depleted state, the amount of amino acids and protein utilized increased and accounted for a higher percent of total energy utilization (Winder et al, 1979). Thus, when carbohydrate is not readily available as an energy substrate, muscle protein may be degraded so that the carbon-chains can be utilized for energy through the oxidation of B C A A (Kasperek and Snider, 1987). Alternatively, the carbon-chains may be used for glucose formation via gluconeogenesis, and subsequent  20  REVIEW OF LITERATURE energy use. For example, in glycogen depleted subjects (resulting from exercise and starvation), it was found that the activity of B C K A D in muscle increased significantly (Kasperek and Snider, 1987). Brouns et al (1989) investigated the carbohydrate (CHO) requirement to adequately glycogen load an athlete so that muscle protein degradation and muscle protein oxidation could be prevented. They fed cyclists who performed 2 days of exhaustive cycling a diet consisting of: a fructose-maltodextrin beverage equal amounts of each component in addition to their normal C H O diet (abbreviated the F M diet); or a normal C H O diet (abbreviated the N diet) in which CHO accounted for 60% of the total amount of energy ingested. The N and F M diets resulted in a negative CHO balance. They found that protein oxidation (as measured from daily nitrogen losses in urine and sweat) significantly increased in the N diet during exercise and recovery and in the F M diet on the second day of exercise, while there was no significant change in protein oxidation in the F M diet on the first day. They suggested that the N and F M diets resulted in a C H O depleted state, which through biochemical changes, lead to increased protein degradation in muscle. On day 1 of the F M diet, the high C H O intake may have counterregulated the biochemical factors inducing protein degradation resulting in no changes in protein degradation. However on day 2, where energy balance remained negative for longer periods of time, protein catabolism may have been initiated. The results of this experiment, however, may be questioned, as the investigators did not measure the amount of protein lost in feces. Duration of exercise may also affect the quantity of muscle protein degradation. It has been suggested that increased exercise duration is associated with increased protein catabolism (Lemon and Mullin, 1980). In fact, it may be that prolonged exercise has the same effects on protein catabolism as starvation (Lemon and Mullin, 1980). Kasperek and Snider (1987) also confirmed the fact that increased exercise duration is associated with increased protein catabolism, especially in muscle. The activation of B C K A D in muscle was elevated as duration of exercise increased resulting in a subsequent  21  REVIEW  OF  LITERATURE  increase in oxidation of B C A A in muscle. Thus, B C K A D is activated in response to increasing energy demands. The variability of muscle protein degradation associated with exercise intensity has recently been documented, where the calpain activity (an enzyme which breaks down Z-line proteins in the myofibril; Johnson, 1990) increased with increasing intensity in the plantaris muscle (which contains fast muscle fibers) of rats (Belcastro et al, 1995). This intensitydependent increase in calpain activity was dependent on the duration of exercise and fiber type, where the intensity effect was not seen when exercise duration was short or when assessed in slow muscle fiber types (the soleus). The effect of exercise intensity and duration on muscle proteolysis has not been documented in humans. Conclusions made about myofibrillar protein degradation during exercise are conflicting. Several investigators have found that muscle protein degradation remained unchanged during exercise, but increased significantly during the recovery period. For example, Dohm et al (1982) found no significant change in the 3-methylhistidine excretion on the first day of exercise involving a 2 hour run. However, during the day after the experiment when no further exercise was performed, the excretion rate of 3-methylhistidine (3-MH) in urine was significantly increased suggesting that contractile protein degradation increased in recovery rather than during the exercise bout. Kasperek et al (1992) suggested that the rate of myofibrillar protein degradation continued into the recovery phase as the urinary excretion of 3-MH was increased for several days after exercise consisting of downgrade running. The fact that myofibrillar protein degradation was unchanged during exercise may represent adaptation of the contracting muscle to prevent degradation of the proteins in the motor units responsible for the continuation of the exercise (Kasperek et al, 1992). Rennie et al (1981) also supported the finding that contractile protein degradation rate is decreased during exercise but is increased during the recovery period if exercise is of high enough intensity and long enough duration. Rennie et al (1981) investigated 3-MH  22  REVIEW  OF  LITERATURE  excretion in urine and 3-MH concentration in plasma of human subjects after running on a treadmill for 2-3.75 hours. There was a fall in 3-MH urine excretion as well as a decrease in intramuscular free 3-MH concentration during exercise. Decombaz et al (1979) also found that 3-MH excretion was not increased during a 100 km running race. Immediately after exercise Dohm et al (1987) found that the production rate of 3-MH increased again. Thus it was suggested that during exercise there was a decrease in the fractional rate of myofibrillar protein degradation and a subsequent increase in myofibrillar protein degradation during the recovery period. However, Kasperek et al (1992) suggested that the large increase in the urinary excretion of 3-MH during recovery from exercise may be due to a decreased food intake. They indirectly observed this by noting that myofibrillar protein degradation was significantly lower in rats that had been starved for 24 hours, were exercised and were refed compared to rats who exercised but were fed at their energy requirement. This demonstrates the profound impact that energy intake may have on muscle protein degradation in exercised animals. Wong and Booth (1990) suggested that protein degradation was increased in the tibialis anterior in rats undergoing stimulatory chronic eccentric exercise. Protein degradation, however, was not measured directly and this suggestion arose from the observation that protein synthesis decreased with repetitive chronic stimulation in the tibialis anterior. Lemon and Mullen (1980) found that protein degradation increased during exercise. They found that urea nitrogen excretion was increased during exercise. However, they stated that the source of protein catabolism could not be determined. Whether one finds an increase, no change, or a decrease in muscle protein degradation depends on the timing and type of measurement made, and the intensity and duration of exercise. Thus, more sophisticated methods are needed before the effects of exercise on muscle protein degradation can be completely understood.  23  REVIEW  OF  LITERATURE  It has been suggested that the increase in muscle protein degradation seen in exercise is related to the loss of structural protein, which subsequently may cause myofibrillar ultrastructural abnormalities (Heyliger et al, 1985). For example, a dramatic decrease in a 58 kDa and 95 kDa protein as well as changes in the ratios of several plantaris myofibrillar proteins were associated with an extensively disrupted sarcomeric structure (Belcastro et al, 1988). It was suggested that these 58 and 95 kDa proteins were probably desmin and actinin (myofibrillar proteins involved with inter and/or intra- Z line structure) (Belcastro et al, 1988). Thus, the loss of protein occurring as a result of increased muscle protein degradation resulted in dramatic ultrastructural changes in the muscle. This increase in muscle protein degradation occurring as a result of exercise may actually represent adaptive changes in protein composition of skeletal muscle occurring with endurance training (Zaidi and Narahara, 1989). For example, increased protein degradation may be needed to cleartissuedebris for deposition of newly synthesized muscle protein during remodeling (Reddy et al, 1983). Recent evidence suggests that protein changes from one isoform to another and that the control site is at the level of transcription (Babif and Booth, 1988). Conclusions made about protein degradation during and after exercise may be conflicting due to the differing exercise protocols and methods of assessing protein degradation. The type, frequency, intensity and duration of exercise was different among studies and may have contributed to the differing results obtained from studies looking at muscle proteolysis and exercise. Additionally, methods of assessing muscle protein degradation may be difficult to interpret and perform (especially during exercise). Furthermore, methods of evaluating muscle proteolysis make it very difficult to determine which myofibrillar proteins are broken down. In summary, a direct relationship between an increase in fat oxidation, induced by an elevated energy expenditure, and an elevated muscle proteolysis has been implied but not established. Some studies suggest that there is an increase in myofibrillar protein  24  REVIEW OF  LITERATURE  degradation during exercise, while others suggest that it increases after exercise. Most of the studies looking at muscle protein breakdown and exercise used athletes as the study population. Therefore, more research is needed to quantify myofibrillar protein degradation in a non-athletic population who is trained.  B) Low Energy Intake A low energy intake in extreme forms (ie starvation) and less extreme forms (ie dieting) may result in increases in myofibrillar protein degradation as a result of the changes in substrate oxidation. For example, prolonged starvation is associated with increased lipolysis (Costill et al, 1977), which in turn may promote changes in myofibrillar protein breakdown, generating glycerol and non-esterified fatty acids. Glycerol acts as a gluconeogenic precursor and non-esterified fatty acids act as an energy source such that there is an increased amount of fatty acid beta-oxidation in this state (Sugden et al, 1989). Therefore the lipid status associated with starvation may promote changes in muscle metabolism. Hickson et al (1977) investigated the effects of starvation on muscle metabolism by using isotopic and 3-methyl histidine (3-MH) excretion measurements. They found that during starvation of rats, muscle protein was not degraded but progressively conserved, until the later stages of starvation. However, Dahlman et al (1986) found that muscle taken from rats during the early days of fasting showed protein degradation. This was evident by a 2-3 fold increase in the amount of easily releasable myofilaments, which are intermediates in the degradative pathway of myofibrillar proteins. Kasperek et al (1992) also found that muscle protein degradation was elevated in rats fasted for 24 hours. This was evident by a significant increase in 3-MH release, measured from skinned hindlimb muscles to eliminate the release of 3-MH from skin into the perfusate. Three-methyl histidine is used as an index of contractile protein degradation because it is not utilized by the muscle (Dohm et al, 1982). Once released, this amino acid can  25  REVIEW OF LITERATURE neither be degraded or re-utilized in de novo protein synthesis and is rapidly removed from the plasma by the kidney and excreted in the urine (Gibson, 1990). There are various limitations associated with using 3-MH to quantify myofibrillar protein degradation. For example, 3-MH release is an actin marker of myofibrillar degradation (Furuno et al, 1990), which represents about 20% of myofibrillar proteins (Vander et al, 1990). As a result, no information can be gained regarding the degradation of other regulatory proteins such as troponin, tropomyosin, or Z-line proteins. Thus, it may not be possible to address issues regarding loss of function since the only structural component accounted for by this method is actin (which does not solely determine muscle function). Furthermore, non-muscle sources of 3-MH have been identified. A small amount of contractile proteins are found in the gut and in the skin and contribute to the urinary loss of 3-MH (Gibson, 1990). Although they represent a small portion of the total 3-MH pool they have a high turnover rate which is faster than in skeletal muscle. The result may be a falsely estimated myofibrillar protein degradation due to the non-muscle contributions of 3M H (Gibson, 1990). Finally, there can also be a problem of interpretation of 3-MH results in situations where muscle wasting occurs (as might be seen with diet restriction). When muscle wasting occurs, the proportion of skeletal muscle diminishes, resulting in a decreased contribution to total 3-MH excretion. Thus, the inability to determine the 3-MH in relation to muscle mass may produce results that may be misleading. The problems associated with the methods of assessing myofibrillar protein degradation make it difficult to make conclusions about muscle protein degradation. More research is needed to compare methods of assessing protein degradation and develop a standardized method of assessing this. Frayn and Maycock (1979) also confirmed early protein degradation with fasting by observing that, in vitro, the extensor digitorum longus of starving rats had a significantly higher rate of protein degradation (3.36 ± 0.21 umol tyrosine released/gram protein, P < 26  REVIEW  OF  LITERATURE  .001) compared to control rats (2.54 ± 0 . 1 umol tyrosine released/gram protein) as measured with [-^tyrosine. These observations, however were dependent on muscle fiber type. There was a significantly higher protein degradation in the extensor digitorum longus, which contains a high number of fast-twitch fibers compared to the soleus, which contains more slow-twitch muscle fibers. These findings suggest that striated muscle protein degradation may play a role in the early response to food deprivation when fat oxidation is enhanced. However, this method of assessing myofibrillar protein degradation does not address the mechanism by which myofibrillar proteins are broken down, or the source from which the released tyrosine came from. Moreover, L i and Wassner (1984) found that contractile proteins and non contractile proteins showed different rates of protein degradation and a different response to fasting. The rates of total protein and actomyosin degradation (representing the contractile protein degradation) were calculated by measuring the release of two non-metabolizable amino acids from the hemicorpus of rats (phenylalanine and N'-methylhistidine). When rats were deprived of food for 48 hours, the rate of total protein degradation increased to 148% of the fed controls while the rate of actomyosin degradation was 47% of that of fed controls. It was found that the contribution of actomyosin breakdown to total muscle protein breakdown was small in the fed state (11%) and increased threefold after food deprivation. In addition, when food deprived rats were refed, there was a decrease in rate of actomyosin degradation to that of the fed controls. These results suggest that a restriction in energy intake may cause a substantial amount of weight loss originating from lean tissues, primarily in the form of water and protein. The change in substrate oxidation occurring as a result of dietary restriction may cause specific proteins, such as contractile proteins to be lost, perhaps by causing a change in the rate of protein degradation and/or synthesis. However, in humans the specific or selective degradation of identifiable, functional protein occurring as a result of a calorie restricted diet has not been examined. Shifts in substrate turnover of resting muscle such  27  REVIEW OF LITERATURE as fasting, unbalanced diets, nervous/hormonal effects, may result in the inclusion of muscle protein/amino acid utilization as an energy source. More research is required to establish a direct cause-effect relationship between fat oxidation and muscle proteolysis as a result of a low energy intake.  5.  PROTEOLYTIC SYSTEMS INVOLVED IN PROTEIN DEGRADATION The proteolytic system(s) that may be involved in the increase in myofibrillar  protein degradation is discussed in this section. Proteins in eukaryotic cells are continually degraded and replaced under precise control mechanisms. Although this continual proteolysis may seem wasteful, it serves several important functions: cells selectively degrade proteins with abnormal sequences or conformations, the accumulation of which could be harmful; rapid degradation of regulatory peptides and enzymes is essential for the control of metabolic pathways and the cell cycle; breakdown of proteins in starvation provides amino acids for gluconeogenesis and energy metabolism; post-translational processing of newly synthesized proteins occurs (Kettelhut et al, 1994). Under conditions of nutritional deprivation and/or hormonal or amino acid deficiency there is a basal rate of proteolysis as well as an additional accelerated rate of proteolysis (Garza et al, 1976). These intracellular proteolytic events are controlled by multiple pathways within mammalian striated muscle cells. Myofibrillar protein breakdown in eukaryotic cells may occur through distinct pathways: lysosomal enzymes; calcium-dependent proteases; ATP-dependent proteases; ATP-independent proteases. The proteases involved in the ATP-independent degradative system is not known (Kettelhut et al, 1994). Only some of these proteases are well characterized, and it was found that they range in size from 20 to 800 kDa (Sugden et al, 1989).  28  REVIEW OF  LITERATURE  Lysosomal proteases are small monomelic units that are compartmentalized within striated muscle (Bond and Bulter, 1987) and include enzymes such as cathespins B, H, and L. They serve in a digestive capacity, reducing peptides into individual amino acid components. They are not in contact with the cellular environment and prefer an acidic pH for optimal activity. Adenosine triphosphate-dependent proteases are high-molecular weight, oligomeric proteins with alkaline pH optimum (Bond and Butler, 1987). The enzymes fall into two distinct classes (Bond and Butler, 1987): one group is found in the cytosol and consists of cysteine proteases; the other group is found in the mitochondria or bacteria and consists of serine proteases. The major differences between the groups lie in the active-site catalytic residue and the mechanism of action of ATP. The cysteine proteases, which do not require magnesium for activity, are allosterically stimulated by ATP (Bond and Buder, 1987). By contrast, serine proteases, which require magnesium for activity, require A T P hydrolysis for proteolytic activity (Bond and Butler, 1987). Protein degradation involving ubiquitin is an ATP-dependent cysteine protease where ubiquitin is first activated in an ATP-dependent fashion and then covalently bound to protein substrates (Attaix et al, 1994). Ubiquitination of proteins always requires the ubiquitin-activating enzyme and one of the ubiquitin carrier proteins to generate the first attachment of a single ubiquitin moiety to a substrate. Substrates can be multiubiquitinated by the attachment of ubiquitin chains or trees (Attaix et al, 1994). Polyubiquitinated proteins are preferentially degraded by a very large 1500 kDa (26 S) proteolytic complex that requires ATP for activation and substrate hydrolysis (Attaix et al, 1994). The 26 S complex contains the 20 S proteasome with proteolytic function and multiple other components. Although the 20 S proteasome has been shown to be an essential component of the ATP-ubiquitin-dependent proteolytic pathway, skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin (Attaix et al, 1994).  29  REVIEW OF  LITERATURE  Non-lysosomal proteases are optimally active at neutral pH. These enzymes, also found in skeletal muscle, are under direct influence of intracellular events (Bond and Bulter, 1987). These enzymes appear to act in more of a regulatory role, instead of a digestive role, as they form restricted fragments from their specific protein substrates in vivo (Bond and Bulter, 1987). The best characterized of the non-lysosomal proteases is a calcium activated neutral protease, calpain (Murachi, 1989). Two active forms of this enzyme can be distinguished by their requirement for calcium: calpain I is half maximally activated by 20-50 uM calcium, while calpain II requires .3-.7 mM calcium for half maximal activity (Li and Wassner, 1984). Calpain is ubiquitous to the cell and selectively degrades myofibrillar proteins, (especially at the level of the Z-line in muscle cells (Brooks, 1987), cytoskeletal proteins, and membrane proteins (Block et al, 1986). Calpains are normally intracellular enzymes (Johnson, 1990). Most intracellular calpain is cytosolic, with between 7-30% of the calpain being associated with membrane structures (Johnson, 1990). Within the membrane, it seems likely that the distribution of calpain is not uniform, but is associated with various membrane components such as membrane phospholipid, structural proteins, transport systems and receptors (Johnson, 1990). Within the sarcomere, calpain I has been found to be associated with the I-band and Z-band region of the sarcomere (Johnson, 1990). Calpain activity in striated muscle is controlled by the following factors (Belcastro et al, 1994): the calcium concentration; autolyzed/unautolyzed state of the enzyme; the level of inhibitor, calpastatin; the availability of digestible substrates. The calpains do not cleave undenatured actin or myosin (Goll et al, 1991). Because these two proteins are the principal sources of 3-MH released during muscle protein degradation, release of 3-MH cannot be used to indicate whether the calpain system has a role in myofibrillar protein degradation (Goll et al, 1991).  30  REVIEW OF  LITERATURE  The calpains do cleave skeletal muscle plasma membrane proteins and phospholipids, as well as cytoskeletal and contractile proteins (Johnson, 1990). The degradation of membrane proteins by calpain (such as band 3 protein, fodrins, and band 4.1 protein) seems to be restricted to the cytoplasmic side of the membrane (Johnson, 1990; Zaidi and Narahara, 1989). Cytoskeletal proteins that are substrates for calpain include (Johnson, 1990): alpha-actinin, tropomyosin, lamins, vimentin, neurofilament protein, tubulin, microtubule-associated protein, C-protein, and desmin. Proteins indirectly involved in myofibril cross-bridge formation can also be degraded by calpain. These proteins include myosin light chain, tropomyosin, troponin-Tlike protein (in smooth muscle), caldesmon, calponin, and alpha-actinin (Johnson, 1990). Their functions involve the maintenance of a myofibrillar conformation needed for crossbridge formation, and the regulation of cross-bridge cycling. For example, alpha-actinin is a protein in the Z-line to which the ends of the F-actin molecule of the thin filaments attach (Murray et al, 1990). Tropomyosin is more of a regulatory protein in that it covers the myosin-binding site on each actin, thereby preventing the cross bridges from making contact with actin (Belcastro, 1988). The binding of calcium produces a change in the shape of troponin such that it pulls the tropomyosin, to which it is bound, out of its blocking position, uncovering the cross-bridge binding sites on actin. It has been suggested that calpain's role in muscle protein degradation is directed exclusively at the myofibrillar or cytoskeletal proteins, where their effects result in disassembly of the myofibril and release of large polypeptide fragments, but not in the liberation of free amino acids (Goll et al, 1991). Thus, it has been suggested that calpain is an ideal enzyme for initiating disassembly of myofibrils (Goll et al, 1991). Zaidi and Narahara (1989) suggest that calpain's role in myofibrillar protein degradation is through the plasma membrane proteins. They found that calpain preparations consisting of plasma membranes from frog skeletal muscle caused a striking loss of a major membrane protein of molecular weight 97 kDa, designated band c.  31  REVIEW OF LITERATURE Overall, the proteolytic systems involved in muscle protein breakdown as a result of an energy restriction may be due to the non-lysosomal enzyme, calpain. Lysosomal enzymes (such as cathepsins) and ATP-dependent proteases (such as those involved in the ubiquitin system) may also be involved. It is difficult to isolate the proteolytic system responsible for the increased myofibrillar protein degradation due to the possible involvement of more than one system, lack of knowledge about the proteolytic substrates, and the lack of methodological procedures quantitating one specific system.  A) Proteolytic Systems Involved in Protein Degradation Occurring with Exercise This section investigates the proteolytic systems responsible for the proteolytic changes in muscle proteins occurring with exercise. Mechanically induced changes in myofibril ultrastructure occurring as a result of prolonged exercise may be an initiating factor for muscle protein degradation during exercise. For example, Friden et al (1983) found that there was extensive myofibrillar Z band streaming and broadening up to 3 days after intense eccentric exercise in human males. In this type of exercise there is a high degree of mechanical stress. Kasperek et al (1985) also found that muscle damage induced by downgrade running was associated with an increased rate of myofibrillar protein degradation (as was shown by an increase in urinary 3-MH release). Warhol et al (1984) also observed post-race ultrastructural changes in runners. They found that all runners showed some degree of myofibrillar lysis, of which damage was confined to individual sarcomeric units. Belcastro et al (1988) found similar results where dissolution of specific Z-line myofibrillar proteins (desmin and actinin as determined by SDS gel electrophoresis) occurred in rats as a result of a running protocol. Belcastro et al (1984) suggested that the alteration in neuromuscular transmission induced by ultrastructural changes causes an uncoupling of excitation-contraction which may provide a favorable cellular environment for the initiation of myofilament degradation.  32  REVIEW OF  LITERATURE  Although the proteolytic system responsible for this is not clear, it has been suggested that the increase in calcium concentration in the cytosol reported following exercise (Dohm et al, 1981) may activate both lysosomal and non-lysosomal proteases. This postulation was based on the observation that the number of lysosomes and the total activity of lysosomal acid hydrolases increases a few days after prolonged exercise. Belcastro (1993) found that level running increased the activity of the nonlysosomal enzyme, calpain, in rats. It was postulated that this was due to an enhanced calcium sensitivity of the enzyme as well as an enhanced susceptibility of myofibrillar substrate protein to calpain action with prolonged level running. The lowered calcium requirement for half maximal calpain activity was thought to be due to an increased ratio of autolyzed to unautolyzed enzyme (the autolyzed forms of u and m calpain were speculated to have a reduced calcium requirement, and an elevated phospholipid concentration (Belcastro et al, 1988). The increase in calcium concentration seen with exercise may activate calpain, which consequently results in changes in skeletal muscle plasma membrane proteins and phospholipids, cytoskeletal and contractile proteins. Disruption of these proteins by calpain during exercise may prevent cross-bridge formation and may decrease the force production during exercise through alterations in plasma membrane proteins (Zaidi and Narahara, 1989) and/or alterations in cytoskeletal proteins (Belcastro, 1988). Friden et al (1983) also suggested that metabolic disturbances during work may have caused lysosomal enzymes to be liberated into the damaged fibers to initiate degradation of myofibrillar material. Beaulaton et al (1977) and Fox (1975) also postulate that the soluble alpha-actinin or Z-line proteinase (activated by the non-lysosomal enzyme calpain) may also be involved in initiating myofibrillar protein breakdown. Other researchers have also concluded that lysosomal enzymes may be responsible for initiating muscle protein breakdown. For example, Dohm et al (1979) found that free cathepsin D activity increased during exercise. They suggested that this enzyme could have  33  REVIEW OF  LITERATURE  leaked out of the lysosome into the cytoplasm and caused an elevated level of muscle protein degradation to occur as a result of exercise. Pilstrom et al (1978) supported this finding by observing that exercise does in fact increase the activity of cathepsin D, as well as the activity of other lysosomal enzymes. Most lysosomal degradation takes place in secondary lysosomes because lysosomal enzymes are largely inactive at the pH values in the cell cytosol. Myofibrils are so large that they could not be assimilated by lysosomes. Therefore, striated muscle myofibrils would have to be disassembled to individual filaments and possibly to filament fragments before the lysosomal cathepsins could have a role in their degradation (Goll et al, 1991). Furthermore, it appears unlikely that large proteolytic complexes such as macropain (the multicatalytic lysosomal protease) could effectively degrade intact myofibrils before they were disassembled into filaments (Goll et al, 1991). It has further been suggested that lysosomal enzymes may not be involved in the increased rate of degradation of contractile muscle proteins but rather that of noncontractile muscle proteins. Kasperek et al (1992) found that chloroquine (an inhibitor of lysosomal fusion) failed to bring the rate of tyrosine release from the isolated soleus muscles of exercised rats to control levels. Thus, it was suggested that the increase in "free" cathepsin D activity observed with exercise caused an increase in the rate of degradation of noncontractile muscle protein (Kasperek et al, 1992). Therefore, evidence suggests that the non-lysosomal system may be responsible for the increase in muscle protein breakdown seen with exercise because the sensitivity of this system is increased and the activity of this system is higher. The effect of exercise intensity, duration, frequency and type on the proteolytic system(s) involved needs to be quantified.  34  REVIEW OF LITERATURE B) Proteolytic System(s) Involved in Protein Degradation Occurring with a Low Energy Intake Both non-lysosomal enzymes and lysosomal enzymes have been implicated in causing proteolytic changes in skeletal muscle with a low energy intake. For example, in one study it was found that calpain activity (a non-lysosomal enzyme) was increased in rats that were calorically restricted in the following manner: 50 % dietary restriction for 2 days; 75% dietary restriction for 2 days; fasted over 2 days (Murray et al, 1991). It was also found that the activity of calpain increased relative to the extent of the dietary restriction: there was a 13% increase in calpain activity with a 50% dietary restriction; there was a 22% increase in activity with a 75% dietary restriction; there was a 29% increase in activity in fasted rats. Thus, calpain may play a pivotal role in muscle protein turnover during a dietary restricted state by initiating the process of myofibrillar disassembly (Murray et al, 1991). The involvement of calpain in increasing myofibrillar protein degradation as a result of fasting comes from a study administering E-64 (a cysteine protease inhibitor that inhibits the calpains). The increase in the amount of easily releasable myofilaments (intermediates in the degradation of myofibrillar proteins (Dahlmann et al, 1986; Van der Westhuyzen et al, 1981) as a result of fasting was reduced by treatment of the animals with E-64. Lysosomal enzymes, such as cathepsin D, have also been implicated in initiating myofibrillar disassembly in a dietary restricted state. For example, in the study by Murray et al (1991) as previously described, it was found that the cathepsin D activity was also significantly increased in dietary restricted groups compared to controls. The extent of this increase, however, was significantly lower than the increase in calpain activity. It has been suggested that calpains, and not the cathepsins, appear to play a role in the enhanced degradation of proteins in energy-depleted muscles (Fagan et al, 1992). Weak bases were administered to ATP-depleted chick skeletal muscles in order to prevent the digestion of proteins with the lysosome by increasing intralysosomal pH. However,  35  REVIEW  OF  LITERATURE  the weak bases did not block the increase in the degradation of proteins in energy-depleted muscles, suggesting that these proteins were degraded by a nonlysosomal proteolytic pathway, such as one involving calpain (Fagen et al, 1992). This was further supported by the fact that 3-MH release did not differ between energy-depleted and control rats when calpain activity was inhibited with E64 (Fagen et al, 1992). This meant that actin and myosin, which are not calpain substrates, were not being broken down (as was indicated by the absence of a change in 3-MH release between energy-depleted and control rats). This suggested that the myofibrillar proteolysis was due to calpain action. Therefore, the proteolytic enzyme, calpain, may have a pivotal role in myofibrillar protein degradation occurring with a low energy intake. More research is required to determine the involvement of other systems as a result of a low energy intake.  6.  MUSCLE  FUNCTION  The purpose of this section is to describe how muscle function changes as a result of increased myofibrillar protein degradation and an energy restriction. A decrease in strength may result in the inability to sustain a required power output. Strength is frequently defined as the peak force or torque developed during a maximal voluntary contraction (Alexander, 1990). Peak strength generation is determined by an optimal and dynamic interaction between the muscle (excitation-contraction processes), neural recruitment (type of motor units, size of motor unit pool, frequency and pattern of discharge), and the metabolic source of ATP production (anaerobic and aerobic; Edgerton et al, 1986). Alterations in muscle protein structure occurring as a result of increased myofibrillar protein degradation may decrease the force generating capabilities of skeletal muscle associated with prolonged repetitive low force producing contractions due to the disruption  36  REVIEW OF  LITERATURE  in sarcomere structure (Edgerton et al, 1986). Specifically, it has been suggested that calpain action on the myofibril produces a loss of muscle function. Goll et al (1991) suggested that the formation of a myofibril with a smaller diameter produced as a result of calpain action, would have less strength and a diminished force production. Belcastro et al (1988) suggest that the changes in relative protein composition of the myofibrillar structure may implicate decrements in force transmission in situations where calpain activity may be enhanced due to the disruption in sarcomere structure (Edgerton et al,1986). Specifically, calpain action on cytoskeletal elements may compromise force generation due to an uncoupling of excitation-contraction (Belcastro et al, 1984) as a result of the impairment in muscle cell endocytosis, exocytosis and intercellular transport (Belcastro, 1988). Zaidi and Narahara (1989) suggest that calpain may compromise skeletal cell muscle function through alterations in sarcolemma structure (for example, changes in membrane fluidity). As a result of the difficulty associated with impulse transmission across the neuromuscular junction, an uncoupling of the excitation-contraction coupling, induced by calpain action, may occur, subsequently resulting in a decreased force transmission. The force transmission failure may be responsible for the presence of fatigue and a decrease in muscle performance during exercise or during continuous training. Associations between myofibrillar protein degradation and muscle function as a result of an energy restriction have not been established. However, energy restriction and athletic performance has been investigated where it has previously been found that an energy restriction combined with excessive exercise may result in a decrease or no change in athletic performance (Beals and Manore, 1994). Ingjer and Sundgot-Borgen (1991) studied the effects of dieting in athletes on V 0  2  max and running speed. The dieting athletes did show a significant decrease in V O ^ max and running speed relative to control during and after 2 months of weight reduction. Thus, there appeared to be an immediate reduction in aerobic capacity and performance. Possible  37  REVIEW OF  LITERATURE  mechanisms for these decreases in muscle function included electrolyte imbalances and cardiovascular changes, glycogen depletion and delayed healing and recovery from injury (Beals and Manore, 1994). The results obtained by Ingjer and Sundgot-Borgen (1991) are in contrast with those found by Scott et al (1992) who found that there was no change in maximal physical performance as a result of an imposed energy restriction. Tliirty-six mildly obese premenopausal females ingested 1000 kcal/day for 8 weeks. The strength values (assessed using a Cybex 340 isokinetic device) did not differ pre- and post-diet. In summary, the effect of an energy restriction on athletic performance yields conflicting results. This may be due to differing diet protocols between studies, differing athletic testing regimens, and differing subject populations. Moreover, an association between myofibrillar proteolysis and muscle function remains to be established.  38  REVIEW OF LITERATURE 7. DIETING AND ATHLETES Female endurance athletes provide a special population for investigation of these issues as they are known to habitually restrict food intake and exercise excessively. Also, although eating disorders have been documented in athletes, relatively little work has been done to examine the physiological effects of a restricted energy intake in athletes. Dieting is known to mean that energy intake is restricted in an attempt to lose weight (Reeds and Fuller, 1983). The fact that female endurance runners ingest a low energy intake has been documented. For example, various studies reported an intake range from 5860 kj/day to 8330 kj/day (1400-1991 kcal/day/) for female endurance runners (Davis and Cowles, 1989). Thus, some female endurance athletes have surprisingly low energy intakes whereas the reported physical performances suggest a high level of energy expenditure. For example, Mulligan and Butterfield (1990) found that female distance runners, averaging 50 km/week, had energy intakes similar to those of female nonrunners (1745 versus 1786 kcal/day for runners), yet the energy expenditure of the runners was significantiy higher than those of non-runners. Moreover, among nonrunners, calculated mean daily energy intake was equal to the calculated energy expenditure but for the runners, calculated mean energy expenditure exceeded mean energy intake by more than 645 kcal/day. Yet all groups were reported as being weight stable over the two month study period. The discrepancy between reported energy intake and energy expenditure may be explained in part by underreporting of food intake (Reeds and Fuller, 1983). None the less, it has been suggested that athletes who are restricting their energy intake may predispose themselves to the development of an eating disorder (Davis and Cowles, 1989). The fact that a low caloric intake may be a problem in the athletic population is demonstrated by the fact that as many as 25 % of athletes may have an eating disorder (Wilmore, 1991). Various factors may predispose an athlete to an eating disorder, or a  39  REVIEW OF  LITERATURE  restricted energy intake. There is a high demand to be thin when an athlete is judged on the basis of how he/she looked during a performance (gymnastics, and diving; Beals and Manore, 1994). There is an increased demand to be thin in endurance sports when a low body weight is equated with a more efficient performance, and therefore a faster time for a given distance (i.e., long distance running) (Wilmore, 1991). It was found that in sports where a thin build was perceived advantageous, there were greater weight and diet concerns, higher emotional lability, and greater dissatisfaction amongst athletes involved in these sports (Davis and Cowles, 1989). Specific psychological characteristics such as perfectionism, goal oriented, and perhaps being under tight control of a parent or coach, may also increase the risk of an athlete becoming involved in restricting energy intake (Wilmore, 1991). The perceived advantage to performance in reducing body weight, as well as the sociocultural demands placed on females to achieve and maintain an ideal body shape, may put intense pressure on the female athlete to diet. In fact, the relationship between dieting and activity may be a vicious cycle that perpetuates itself: activity may promote dieting, which in turn induces activity (especially during the early stages of food restriction) (Davis and Cowles, 1989). For example, strenuous exercise may suppress appetite as a result of changes in endorphin levels (Katz, 1986), which serves to decrease the value of food reinforcement. As a result, food intake decreases and body weight is lost. As body weight decreases, the motivation for more exercise increases. Thus, it has been suggested that the increased training load may have induced a caloric deprivation in endurance athletes, which in turn has elicited certain biological and social reinforcements leading to the development of an eating disorder (Sundgot-Borgen, 1994). Weight preoccupation in the form of dieting can represent one of the progressive steps to a more severe form of weight preoccupation such as anorexia nervosa and/or bulimia in some female athletes (Beals and Manore, 1994). Weight preoccupation often  40  REVIEW OF  LITERATURE  coincides with restrictive eating behaviour. Thus, the physiological effects of dieting in athletes was studied. The effects of a low energy intake in athletes may be enhanced due to possible additive effects of low calorie intake and increased energy expenditure on muscle protein degradation. Changes in muscle protein degradation, induced by increased fat utilization, may decrease total muscle mass and fat mass, and therefore decrease performance (Beals and Manore, 1994).  8.  SUMMARY Overall there has been extensive research on exercise as it is related to myofibrillar  protein degradation, or a low energy intake as it is related to myofibrillar protein degradation. However, few studies have actually looked at the combined effects of an exercise program and a low energy intake on myofibrillar protein degradation. Furthermore, most studies have looked at the combined effects of diet and exercise on myofibrillar protein degradation in obese subjects. Information about the mechanism by which myofibrils are broken down as a result of an energy restriction is limited. In conclusion, it is evident that more research is required to determine if the increase in fat oxidation is responsible for the proteolytic changes in muscle proteins seen with an energy restriction. Additionally, the activation of calpain as a result of this change in substrate oxidation remains to be established. Subsequently, the changes in myofibrillar protein breakdown as a result of calpain action may have detrimental effects on muscle function. A relationship between muscle function and calpain activity has not been previously determined. Therefore, more information regarding the effect of an energy restriction in the form of diet and exercise is needed to further understand the relationship between substrate oxidation, calpain activity, and muscle function.  41  EXPERIMENTAL  DESIGN  Chapter III  E X P E R I M E N T A L DESIGN /.  STUDY  DESIGN  The basic design of the study was that of an intervention study extending over approximately a two week period. The hypotheses were tested in male and female subjects. Male and female subjects were provided with all of their meals for the study duration, and all exercise was supervised and/or monitored in the male subjects. Exercise was performed as usual in the female subjects. Experimental male subjects received nutrition and exercise intervention (i.e. 2/3 of the energy deficit arose from dietary restriction, 1/3 of the energy deficit came from increased energy expenditure). Female subjects received nutrition intervention only; the imposed deficit in these subjects arose solely from a dietary restriction. All male and female subjects ingested breakfast and lunch at the Family and Nutritional Sciences Building, University of British Columbia. Dinner and a snack were prepared and packaged and were taken home by the subjects. All food was weighed and measured to provide accurate amounts to meet the diet protocol and individual requirements. The three-day rotating menu followed for male and female subjects is shown in Appendix 1. All individual menus were calculated from this master menu. For example, to determine the amounts of food for a person who required 3000 kcal/day, values in the master menu were multiplied by a proportional factor to obtain the correct dietary intake amounts. The dietary analysis of this menu is shown in Appendix 2.  42  EXPERIMENTAL  DESIGN  Thus, the average percent protein, carbohydrate and fat for all three days was 15 ± 2,56 ± 3, and 28 ± 2% respectively, which were within the recommended limits suggested for a healthy diet (Hunt and Groff, 1990). Energy intakes for male and female subjects were determined by estimating resting energy expenditure (REE) through the Harris-Benedict equation (Harris and Benedict, 1919), and multiplying REE by an activity factor to determine total energy expenditure (TEE):  For men:  R E E = (kcal/day) = 66 + (13.8 X weight in kg) +(5.0 X height in cm) - (6.8 X age in yrs)  For women:  R E E (kcal/day) = 665+ (9.6 X weight in kg) + (1.8 X height in cm) - (4.7 X age in years)  T E E Occal/day) = REE x activity factor. The thermic effect of food was not accounted for in these equations. For the male subjects, total energy expenditure was calculated by multiplying the R E E by an activity factor (1.55, for light activity; Harris and Benedict, 1919). For the female subjects, total energy expenditure was calculated by multiplying the R E E by an activity factor (1.82, for heavy activity; Harris and Benedict, 1919). The activity level of each female subject was determined in the initial interview by discussing with the subject how much and what type of exercise she did on a regular basis.  43  EXPERIMENTAL  DESIGN  A) MALES Young healthy male subjects were recruited. The energy requirement of the experimental subjects was cross-checked with the aid of a four day diet record, after which they received nutrition and exercise intervention resulting in a 33% energy deficit A series of baseline measurements were taken 3 days prior to the period of diet and exercise manipulation, as well as 1 to 3 days after the period of diet and exercise manipulation. These included anthropometric measures of height (ht), weight (wt), and skinfolds at four sites (triceps, biceps, subscapular, suprailiac). Indirect calorimetry was used to assess substrate oxidation. To evaluate muscle function and assessment of quadricep muscle group strength, a Kin-com isokinetic dynamometer was performed. To assess alterations in muscle protein degradation, human myofibrillar preparations (obtained from a muscle biopsy) were subjected to calpain digestion. Exercise prescription involved daily walking/running on a track until 11% of the total energy requirement had been expended (about 30 minutes of walking/jogging). Measurement of V C ^ max, implemented with a cycle ergometer, and heart rate response to a known workload, were used to determine the amount of energy expended during exercise. A male control group, originating from a previous study (Parkes et al, 1994), were placed on a 100% and 67% of maintenance energy requirement for a time period identical to this study. The control males, however, were completely sedentary. Thus, the 33% deficit originated solely from a dietary deficit  B) FEMALES Female long distance runners were recruited for both the control and experimental groups. The energy requirement of the subjects was assessed with the aid of a four-day diet record and the Harris-Benedict equation, after which the experimental group received nutrition intervention resulting in a 25% caloric deficit for a 10 day period. All subjects ingested a caloric intake at their energy requirement two to four days prior to the 10 day diet  44  _ _  EXPERIMENTAL  DESIGN  period and weight was monitored to determine if the energy level was appropriate. If a subject lost or gained weight while ingesting their calculated maintenance energy requirement, the caloric intake was adjusted before the initiation of the dietary protocol. Before candidates began the diet protocol they completed: a V O ^ max test; an anthropometric assessment (which included height, weight, and 7 skinfolds); a thorough description of their present exercise status and medication history. If the candidate met the criteria of the study the following initial measurements were done: Kin-Corn muscle function test, muscle biopsy, substrate oxidation measurement. Subjects were then randomly assigned to the control or experimental group, after which they ate their respective diets for 10 days. During the dietary period, all subjects were asked to keep a precise exercise diary. An example of a daily exercise recording form can be seen in Appendix 3. At the end of the dietary period the following final measurements were made: Kin-Corn muscle function test; muscle biopsy; anthropometric indices; substrate oxidation.  2.  SUBJECTS A) MALES Eight male subjects were recruited from the University area. Subjects were  recruited using posted notices describing the study's objective and the benefits it would provide for the participants. An example of a recruitment advertisement can be see in Appendix 4. Subject criteria included: - males - non-smokers - free from illness and requiring no medication - body fat content between 1 0 % and 2 0 % - previously sedentary (previous exercise should have been no longer than one hour per week)  45  EXPERIMENTAL  DESIGN  - no consumption of extreme diets such as very high or very low fat diets (greater than 45% and less than 20% of energy intake respectively). - no food allergies.  B) FEMALES Fourteen women were recruited to participate in the study. Subjects were recruited using posted notices describing the study's objective and the benefits it would provide for the participants. An example of a recruitment advertisement can be see in Appendix 5. The total sample size recruited was 14 women, of which 7 were in the experimental group, and 7 were in the control group. This sample size was chosen based on the subject number (n = 8) used in a different study of similar design, where significant differences were found in muscle function as a result of an imposed energy restriction in male subjects (Parkes et al, 1994). A sample size was not calculated because the variability associated with calpain activity in the subject types studied has not previously been documented. The fourteen elite female athletes who were recruited were defined as those who possessed the following criteria: - taking oral contraceptives and/or having a regular menstrual cycle (every month for the past two years)* - not taking anti-inflammatories - body fat between 16-26% - long distance runners - distance run per week at least 50 km - VC»2 max greater than 42 ml/min/kg on a cycle ergometer**  46  EXPERIMENTAL  DESIGN  T h i s criteria was specified because recruitment of subjects with similar hormonal profiles was desired, as hormones have been shown to have an effect on muscle protein degradation (Goldfarb and Kendrick, 1981).  **This VC»2 max value was chosen because it is considered to be an excellent value for women between the ages of 20-29 years (Canadian Standardized Test of Fitness Operations Manual, 1986), and therefore would reflect women who are well-trained. Exclusion criteria for the study included those candidates who possessed food allergies and/or intolerances. These subjects were omitted from the study due to the necessity to keep the menu similar among subjects. All male and female subjects provided written informed consent prior to participating in the study (see Appendices 6 and 7 respectively).  47  EXPERIMENTAL  3.  STUDY  DESIGN  PROTOCOL  Figure 2: Study protocol.  Measurement Anthropo-metrics Substrate Oxidation Muscle Biopsy Muscle Function V02 Max  DAYS A B -3 -2 -1 0  I to 4  C 5  to 11 12 13 14  X  X  X  X X  X  X  X  X  A: Pre-diet phase B: Maintenance diet phase C: Experimental diet phase  48  METHODS  METHODS  1. DIETARY  INTAKE  To assess "usual intake" subjects were instructed to record the type and amount of all foods, beverages (including snacks), and dietary supplements consumed on a dietary intake form for a four day time period (which included at least one weekend day). Subjects were encouraged to maintain their normal eating patterns as much as possible because it has been suggested that recording the amount of food consumed may affect the actual intake in an attempt to circumvent inconvenient or complex entries (Recker et al, 1992). Food intake studies have suggested that women may ingest extra energy in the luteal phase of the menstrual cycle as compared to the follicular phase (Dalvit, 1982; Lyons et al, 1989). Thus, to maintain some consistency among women regarding the ingestion of food during different phases of the menstrual cycle, the female subjects were asked to record their food ingested for four days during the follicular phase of their menstrual cycle. The male subjects were simply asked to submit one 4-day diet record before the 10 day diet and exercise manipulation period. The subjects were asked to give thorough descriptions, including brand names (Gibson, 1987), of all foods and beverages ingested. Measurement of the amount of food eaten was encouraged by using weights, standard household measuring cups, spoons, and rulers for foods such as meat and cake, and counts for other food like eggs and slices of bread (Gibson, 1987). A copy of dietary record instructions provided to subjects is available in Appendix 8. The food intake record form (Appendix 9) was modified from the U B C Hospital, U B C Site, Dietary Department, Dietary History Food Intake Form and the food intake form developed for a previous clinical nutrition study (Mertz and Kelsay, 1984). For a small group, a 4-day food record has been shown to determine usual protein intake within 10%  49  METHODS with 95% confidence (Basiotis et al, 1987). A minimum of three days of food intake records are required for estimating usual energy intakes (Basiotis et al, 1987). Completed food records were reviewed with the subjects. Portion size measures were analyzed for energy and nutrient composition using the computer program Food Processor U (enhanced version 3.14, E S H A Research, Salem OR). Use of the Canadian Nutrient File database allowed for a more accurate representation of the subjects food composition intake (ESHA Research, enhanced version 3.14, E S H A Research, Salem OR). Information regarding foods not available in Canada were obtained from the E S H A database. The Canadian Nutrient File has been shown to be deficient for certain nutrients that have been shown to be underestimated. The American (ESHA) database is substantially more thorough in this respect (Barr et al, 1994). The average daily values were determined from the four day records. The intake of the following dietary components were calculated for foods ingested in this study: calories, carbohydrate, protein, total fat, saturated fat, polyunsaturated fat, monounsaturated fat, cholesterol, dietary fibre, calcium, copper, iron, magnesium, potassium, phosphorus, zinc, sodium, pantothenic acid, thiamin (Bp, B  riboflavin (B2), niacin (B^), vitamin Bg, vitamin  vitamin A , and vitamin C. All dietary intakes were analyzed by one investigator to reduce variability in  translation of intakes.  2.  ANTHROPOMETRICS A) WEIGHT All subjects were weighed on a medical balance scale, which is accurate to the  nearest 0.1 kg. The scale was calibrated to zero between subject weighings (Dikovics, 1987). All subjects wore minimal clothing (no shoes, shorts and t-shirts) for each measurement to minimize variability (Dikovics, 1987). Subjects were asked to stand in the center of the platform, with their arms at their side. All subjects were weighed in the  50  METHODS morning, in a fasted state. However, it was not always possible to weigh subjects before they had consumed fluids and/or emptied their bladder (Dikovics, 1987). Body weight of all subjects was measured before the test period, daily during the test period, and after the test period. Resulting body weights were compared to ideal body weight according to Metropolitan Height and Weight Tables 1983 (Metropolitan Insurance Company; refer to Appendix 10).  B) HEIGHT Height was measured using a stadiometer, which was read to the nearest 0.1 cm (Health Promotion Directorate, 1988). Height was measured twice throughout the duration of each study phase: at the beginning and at the end. For each measurement, heights were measured twice. If the heights were significantly inconsistent, a third measurement was made, and the two most similar heights averaged (Dikovics, 1987). The measurement was done with the subject barefoot in the center of the platform with feet parallel and heels together. Their back was kept as straight as possible with their shoulders, buttocks, and heels in contact with the vertical surface. The arms were left hanging comfortably at their sides, and the head was kept erect, with the Frankfort plane horizontal (the line from the lower orbit to the upper external auditory meatus). The horizontal bar was then lowered at an angle of 90 degrees making contact with the head (Dikovics, 1987). See Appendix 11.  C) BODY MASS INDEX (BMI) Body mass index was calculated from the averaged weight and height measurements using the following formula: BMI = Weight (kg) Height (m) 2  51  METHODS  D) SKINFOLDS All measurements were made with Lange calipers (Cambridge Scientific Industries Inc., Cambridge MD) to indirectly assess body fat. All precision calipers are designed to have a standard contact surface area of 20 to 40 millimeters squared and exert a defined and constant pressure of 10 grams per square millimeter throughout the range of measured skinfolds. Skinfold measurements were made in triplicate. Actual skinfold values were recorded to 0.5 mm. An average of the three values was used, and if one value varied by more than 10% of the others, it was disregarded and a fourth measure taken (Dikovics, 1987). The specific location of each skinfold thickness measurement for male and female subjects was kept consistent to ensure reliable results (Dikovics, 1987; Gibson, 1987) by marking the site with a pen. For all measurements, the caliper was applied for three seconds before the reading was made. To eliminate inter-examiner error, all of the skinfold measurements were taken by the same examiner throughout the study. All measurements were made on the right side of the body (Lohman et al, 1988).  i) M A L E S Skinfold measurements were taken at four sites (triceps, biceps, subscapular, suprailiac). Percent body fat was estimated from the sum of the four skinfolds using tables published by Durnin and Womersley (1974; refer to Appendix 12). The triceps skinfold was taken 1 cm from the midpoint of the upper left arm, between the tip of the olecranon and the acromion process, with the arm hanging relaxed as seen in Appendix 11 (Lohman et al, 1988). A vertical pinch of skin and subcutaneous fat was pulled away using the thumb and index finger directed inferiorly. In order to make certain that no muscle was included in the skinfold, the subjects flexed their triceps. For the measurement of the biceps skinfold thickness, a vertical fold was raised on the anterior aspect of the arm, over the belly of the biceps muscle. This was done 1 cm  52  METHODS superior to the line marked for the measurement of the triceps skinfold thickness on a vertical line joining the anterior border of the acromion and the center of the antecubital fossa. The subject stood facing the examiner, with the arms relaxed at the side, and the palm directed anteriorly. A vertical skinfold was then raised with the left hand and its thickness measured to the nearest 0.5 mm. The calipers were applied for three seconds before the reading was made, followed by the release of the calipers and of the fingers, and the immediate recording of the values (Dikovics, 1987; Lohman et al, 1987). The suprailiac skinfold was measured in the midaxillary line superior to the iliac crest. The subjects were asked to stand in an erect position and to place their arms at their sides with their feet together. An oblique skinfold (aligned inferomedially at 45 degrees to the horizontal) was grasped just posteriorly to the midaxillary line following the natural cleavage lines of the skin. The caliper jaws were applied about 1 cm from the fingers holding the skinfold, and the thickness was recorded to the nearest 0.5 mm (refer to Appendix 11). The measurement of the subscapular skinfold was taken at a locale that was just beneath the inferior angle of the scapula. The subscapular skinfold was grasped on a diagonal, inclined infero-laterally approximately 45 degrees to the horizontal plane in the natural cleavage lines of the skin. The subject was asked to stand erect, with the upper extremities relaxed at the sides of the body. To locate the site, the examiner palpated the scapula with her fingers along the vertebral border of the scapula until the inferior angle of the scapula was identified. For some subjects, the inferior angle was identified by gently placing the subject's arm behind the back. The caliper jaws were applied 1 cm inferolateral to the thumb and finger raising the skinfold, and the thickness was recorded to the nearest 0.5 mm (refer to Appendix 11). Refer to Appendix 13 for data collection sheet used for the male subjects.  53  METHODS ii) F E M A L E S Skinfold measurements were made at seven sites to utilize the female specific equation to determine body fat. Seven skinfold measurements were completed at the beginning and end of the dietary period for all subjects and included: abdominal, chest, axilla, triceps, subscapular, suprailiac, front thigh. The sites for the triceps, subscapular, and suprailiac skinfold measurements were the same as those described for male subjects. The abdominal skinfold was taken one centimeter inferior to and three centimeters lateral to the midpoint of the umbilicus. All measures were made to the right of the umbilicus so that consistency could be sustained. For the measurement, subjects were encouraged to breathe normally and relax their abdominal muscles as much as possible. A horizontal skinfold was then taken with the left hand and its thickness measured to the nearest 0.5 mm. The axilla skinfold was measured at the level of the xiphi-sternal junction, in the midaxillary line, with the skinfold horizontal. The subject was asked to stand erect and slightly abduct and flex the arm at the shoulder joint.  The skinfold was taken horizontally  with the left hand and measured to the nearest 0.5 mm (refer to Appendix 11). The chest skinfold was measured using a skinfold with its long axis directed to the nipple. The thickness was measured 1 cm inferior to the anterior axillary fold. The measurement was made to the nearest 0.5 mm while the subject stood with her arms hanging relaxed at their side (refer to Appendix 11). The thigh skinfold was measured in the midline of the anterior aspect of the thigh, midway between the inguinal crease and the proximal border of the patella. In order to locate the inguinal crease, the subject was asked to flex the hip. The subject then extended the leg so that the distal reference point, located on the proximal border of the patella, could be located. A vertical skinfold thickness was measured while the subject was in a standing position. For the measurement, the subject was asked to shift body weight to the other foot and to relax the leg on which the measurement was made while keeping the right knee  54  METHODS slightly flexed and the right foot flat on the floor. If maintaining balance was a problem, the subject was asked to hold on to a counter top. The caliper jaws were applied about 1 cm distal to the fingers holding the fold and the skinfold was measured and recorded to the nearest 0.5 mm (Lohman et al, 1987; refer to Appendix 11). Refer to Appendix 14 for the data collection sheet used for female subjects. Total body fat was determined from skinfold measurements by first calculating body density (using a population specific regression equation), which was then used to determine percent body fat (using an empirical equation). Subsequently, total body fat was determined. Body density (BD) was calculated using a generalized regression equation derived for women varying in age and body composition (Jackson et al, 1980; see below). This equation showed that multiple correlations for body density equations and the sum of seven skinfolds was 0.852 with a standard error of 3.8% body fat (Jackson et al, 1980).  B D = 1.0907 - 0.0004697 l(Xj) + 0.00000056(X ) - 0.00012828(age) 2  1  3 where B D is in units of kg/m ; X ^ = the sum of all seven skinfolds in millimeters (Jackson et al, 1980).  % Body Fat (%BF) = (495/BD) - 450  (Jackson et al, 1980)  Total body fat = (Body Weight (kg) x %BF)/100  (Gibson, 1990))  The fat-free mass (FFM) for both male and female subjects before and after the study period was also determined:  Fat-free mass (kg) = Body weight (kg) - Fat mass (kg)  55  METHODS The FFM is the mass of the body excluding all extractable lipid (Mitchell and Truswell, 1987). Thus, FFM does not include the "essential" lipid component of the body.  3. EXERCISE  INTERVENTION  A) EXERCISE PROTOCOL i) MALES After completion of baseline measurements, the male subjects participated in a 10 day moderate intensity endurance exercise regimen, in which all subjects exercised for 9-10 days. Exercise sessions were organized so that all subjects were able to participate for at least 9 days. However, if a subject was not able to make the exercise session, he was asked to make up for the missed session on his own time, and make the appropriate heart rate recordings. The exercise protocol involved a 5 minute period of warm-up stretching led by the exercise supervisor, followed by a 25 minute run/walk at 60-80% of each individual's maximum heart rate (as determined by the VC«2 max test on a cycle ergometer). This was followed by a 5 minute cool down, consisting of walking and stretching. Subjects were educated about their heart rate range and the recording of their heart rates throughout the exercise period. They were instructed to keep their heart rate in a certain range during the 20-25 minute run/walk for the dual purpose of ensuring that the subjects were exercising at a high enough intensity and to calculate the amount of energy expended during the exercise period. Heart rates were measured by the subjects themselves as called out by the exercise supervisor and recorded by the exercise supervisor every 5 minutes.  56  METHODS ii) F E M A L E S The female subjects, who received no exercise intervention, were instructed to exercise during the 10 day testing period as they regularly would. The female subjects recorded all exercise sessions on an exercise recording form shown in Appendix 3. The time spent running and the intensity at which the subjects ran at was specified. For all other activities, subjects were instructed to specify the amount of time spent exercising, and the intenisty of the workout.  r B) ENERGY  EXPENDITURE  DETERMINATION  i) M A L E S Before starting the exercise intervention period, a VC«2 max test was done for male subjects. During this test, subjects cycled on an electronically braked cycle ergometer (Mijnhardt, Mode KEM-3, Bunnik, Holland, 1987) for 15-20 minutes at the Allan McGavin Sports Medicine Clinic at UBC, with a 5 minute warm-up, a 10-12 minute period of increasing work load, and a 5 minute cool-down. Heart rate was monitored every 15 seconds throughout the duration of this test. Based on each subjects performance, an equation relating oxygen consumption (dependent variable) and heart rate (independent variable) was obtained, as the oxygen consumption and heart rate are directly proportional to one another (Sharkey, 1990). The regression equation provided a value for oxygen consumption for each 5 minute period. For equations of all subjects, refer to Appendix 15. The total oxygen consumption occurring during an exercise session was determined from the sum of all of the 5 minute oxygen consumption values. This value was then multiplied by the caloric equivalent of oxygen, which is assumed to be 5 kcal/L oxygen during exercise exceeding 70% of the aerobic capacity (Fox et al, 1993), to obtain the amount of energy expended during the exercise session (see equation below). It was assumed that a heart rate obtained at the end of each 5 minute period was a representative value for the entire 5 minutes of exercise.  57  _  _  METHODS  Energy Expenditure (Kcal) = Oxygen consumed (1730 min) x 5 Kcal/L oxygen  ii) F E M A L E S The amount of energy expended by the female subjects was calculated using the Weight Loss Programmer (Version 3.1, Ohio Distinctive Software, Cleveland, Ohio, 1992). In order to calculate the amount of energy expended by running, the time spent running and the intensity of the workout was entered into the computer, and the program computed the energy expended. Determination of the energy expended for activites other than running was calculated in the same manner. However, two subjects forgot to specify the time spent exercising for activities other than running for two and three days respectively when recording their exercise time, and simply checked off the time period in which they were active. Thus, in order to calculate the energy expenditure from these subjects, the lowest time point in the range of time checked off was appointed as the time spent exercising. For example, if a subject checked off the 31-60 minute time range, then the exercise time entered into the computer was 31 minutes. The time spent exercising was also computed from the exercise recording forms by addition of the time spent exercising for each different activity.  4.  SUBSTRATE  OXIDATION  Substrate oxidation was evaluated by determining the resting energy expenditure (REE), the resting respiratory quotient (RQ), and post-prandial RQs using indirect calorimetry. Carbon dioxide production ( V C O 2 ) , oxygen consumption (  V0 ), 2  R E E , and  RQ were determined by using the Sensormedics M M C Delta Trac metabolic cart (Summit Technologies, Anaheim, Ca) located in the Nutrition Laboratory, School of Family and Nutritional Sciences.  58  METHODS Subjects reported to the Family and Nutritional Sciences Building early in the morning. They were instructed not to consume any food or beverages or engage in any physical activity before the testing period. Upon arrival, they were asked to lay in a supine position for 30 minutes and rest, after which R E E and RQ were measured. Measurements were done in a quiet, dimly lit room at a constant temperature. While lying supine on a bed, subjects were asked to relax and close their eyes without sleeping. If the subject became cold, a blanket was available. Relaxation tapes were also available to the subjects to help them relax. Resting energy expenditure and RQ were measured for 20-30 minutes (determined by the consistency of the values) at the beginning and the end of the experimental manipulation period for both male and female subjects. Values were considered consistent: when they did not change from one measurement to the next (the values were within 35 kcal/day of one another). The equipment that was used is summarized in Appendix 16. The subject's head was enclosed in a plastic canopy with a flexible seal at the neck. A canopy system is useful in preventing discomfort associated with nose clips and mouth pieces. The subject inhaled room air through an opening in the canopy. Exhaled air was drawn by a slight suction at a constant low rate to the metabolic monitor mixing chamber where it was analyzed for oxygen and carbon dioxide. Resting energy expenditure and RQ were monitored for approximately two and a half hours after a test breakfast was ingested by the subject. The test breakfast provided 30% of the maintenance energy intake for all subjects pre-diet, and 30% of the daily energy intake of the respective diet group post-diet. The breakfast consisted of 12% protein, 60% carbohydrate, and 28% fat (refer to breakfast ingested on day two of the menu in Appendix 1). Thirty minutes after the breakfast was consumed, the first five minute measurement was taken, after which a five minute measurement was made every thirty minutes four  59  METHODS more times. Results from McCargar et al (1989) suggest that this schedule of data collection was appropriate for the proposed REE and RQ in this experiment. An attempt was made to mease the substrate oxidation measurements for the female subjects during the follicular phase of the menstrual cycle, as it has been shown that resting energy expenditure is elevated in the periovulatory phase of the menstrual cycle (Herring et al, 1992). The follicular phase of the menstrual cycle was defined as the 15 days following the initiation of menstruation (Fox et al, 1988). However, due to difficulties associated with scheduling subjects for testing times, substrate oxidation measurements were not made in the follicular phase for three subjects.  A) RESTING ENERGY EXPENDITURE Resting energy expenditure (REE) is the energy necessary to maintain the physiological systems at rest (Van Zant, 1992). The following equation was used to determine REE: R E E = [(3.941 x V 0 ) + (1.106 x V C 0 ) ] x 1.44 (Weir, 1949) 2  Where V 0  2  2  and VCC» represent the volume with respect to the milliliters of 2  oxygen inspired and carbon dioxide expired, respectively; 1.44 is the multiplier required to extrapolate the values over 24 hours. R E E may account for 60-75% of the daily energy expenditure and is closely correlated to the fat-free body mass (Van Zant, 1992). Therefore, in addition to presenting R E E in terms of its absolute value, it was also expressed in terms of F F M and body weight. The thermic effect of food may also contribute to the resting energy expenditure as the thermic effect of food has been shown to last many hours (Mole', 1990).  B) RESPIRATORY QUOTIENT The respiratory quotient (RQ) is equal to the volume of carbon dioxide produced divided by the volume of oxygen consumed (Hunt and Groff, 1990). The R Q can provide information regarding the type of fuel utilized for energy. For example, when carbohydrate  60  METHODS is being oxidized the RQ is 1.0. When fat is being oxidized, the R Q is 0.70 (Hunt and Groff, 1990). Determination of the R Q for protein oxidation is more complicated because metabolic oxidation of amino acids involves removal of the nitrogen, some oxygen, and carbon as urea. Thus, the R Q for protein varies according to the type of protein being oxidized. The R Q for a typical mixed diet is considered to be about 0.82 (Hunt and Groff, 1990). A relatively accurate non-protein RQ was obtained even though urinary nitrogen losses were not measured due to the fact that the Delta Trac indirect calorimeter assumed that 13 grams of nitrogen were lost each day. Thus, although an exact value for individual nitrogen losses was not available, an attempt was made to account for oxidation of proteins.  5.  MUSCLE  FIBER  ANALYSIS:  A muscle biopsy was taken at the beginning and end of each experiment. The site chosen for the biopsy was the vastus lateralis muscle because this muscle is used significantly during running. The skin around this area of the thigh was first sterilized, after which the skin and subcutaneous tissue was infiltrated with 7 to 10 ml of 2% lidocane without epinephrine. A 5 to 7 mm stab incision was made on the anterolateral surface of the thigh at the junction of its midal and distal thirds. This position was selected because it is least likely to result in significant complications (Kirby et al, 1982). The biopsy needle was inserted perpendicular to the skin surface and advanced 2 to 5 cm into the vastus lateralis to the femur with the needle window closed facing posterolaterally. Withdrawing the inner cutting needle 1 to 2 cm opened the window. This procedure provided sufficient tissue to perform the required protein isolations and resulted in minimal complications (Kirby et al, 1982).  61  METHODS The muscle sample was then removed and blotted to remove surface blood after the needle was withdrawn. Adjacent fat and connective tissue was then dissected free and the remaining tissue weighed and frozen in pre-cooled isopentane stored in liquid nitrogen. Pressure was applied to the wound until bleeding stopped. Sterile adhesive strips and an anchor dressing were then applied to the edges of the wound. Male and female subjects had 2 muscle biopsies performed: one occurred prior to the testing period, and another occurred on day 9 or 10 of the testing period. After the muscle biopsy was taken, the calpain activity of the resulting muscle fibers was assessed. In preparation for these assays, the muscle fibers were homogenized in a borateKC1 buffer (pH 7.0) containing 39 mM sodium borate, 25 mM KC1 and 5 m M E G T A (Belcastro et al, 1991). The homogenate was then centrifuged at 13,000 rpm for 15 minutes (Hermle 360Z, rotor VO2805) and the supernatant, which represented the soluble particulate fraction, was transferred to an eppendorf tube and placed on ice. The soluble and particulate fractions were then separated. This involved washing the pelleted material with 0.33% Triton-X 100 for removal of membrane bound proteins (while maintaining calcium-dependent proteolytic activity). The pellet was homogenized and centrifuged again under the same conditions as before. The supernatant, which represented the particulate bound fraction (membranous calpain), was placed on ice.  62  METHODS  The samples were then prepared for the assay: Table 1: Table of reagents to add in determining myofibrillar calpain activity.  CaCl2§  DDH2O  Buffer 1  Sample  Casein*  Tube  (ul)  (ul)  (ul)  (ul)  (ul)  Blank  200  0  100  0  75  0  200  100  0  75  0  200  100  75  0  0  200  100  0  75  0  200  100  75  0  Eppendorf  Soluble unactivated Soluble activated Bound unactivated Bound activated  *Concentration o f stock solution = 50 mM. C o n c e n t r a t i o n o f stock solution =10  mg/ml.  As soon as the sample was added, the eppendorf tubes were vortexed and incubated at 30 degrees Celsius in a drybath for 30 minutes. After 30 minutes of incubation, the protein assay was performed. This involved putting 100 ul of sample solution in the appropriate wells on the microplate, after which the sample solutions and the blank were read in the microplate reader. Each sample/fraction was done in triplicate.  6. MUSCLE FUNCTION ASSESSMENT Muscle function assessment was accomplished by determination of quadriceps muscular strength. This was done with four knee extensions and flexions using the KinCom isokinetic dynamometer (Kin-Com: computerized exercise, testing, and research  63  METHODS  system, Model 5030, Med-ex Diagnostics of Canada Inc., Port Coquitlam, B.C., 1985). This method has previously demonstrated a high correlation between strength (determined on the isokinetic dynamometer) and athletic performance. For example, a correlation coefficient of 0.87 was found between peak total leg strength per unit body weight and the 100-yd sprint times of a women's track team (Komi, 1986). Strength is frequently defined as the peak force or torque developed during a maximal voluntary contraction (Alexander, 1990). Isokinetic dynamometers measure the muscle moment, which is the muscle force application times the length of the radius of the lever arm from the axis to the line of muscle pull, which is usually labelled the "muscle torque". Thus, torque scores are actually the moments of force produced by muscles contracting to produce rotation around a joint (Alexander, 1990). In order to ensure that the radius of the lever from the axis to the line of muscle pull was consistent between subjects, the bottom of the calf pad of the lever arm was placed at a distance that was 75% of the distance between the head of the fibula and the lateral malleolus for each subject. In order to calculate torque, the distance between the point of application of the generated force and the axis of rotation of the exercise arm was entered into the computer (which quantified the amount of force exerted on the calf pad of the Kin-Com). Recruitment of a homogenous sample of male and female subjects suggested that the neural recruitment required for peak strength generation did not differ significantly between subjects of each gender. Furthermore, the fact that the female athletes were endurance athletes and the male subjects had not been involved in regular exercise for the previous two years suggests that the energy pathways required to reach peak strength (phosphocreatine, glycolytic) using the Kin-Com are not compromised. In the female athletes, these pathways were replete because they are minor pathways used in the type of training in which the female subjects were involved (Fox, 1984) and therefore are unlikely to be a confounding variable. The energy stores were also replete in the male subject group due to their inactivity pre-treatment, and utilization of aerobic training during the study.  64  METHODS Thus, the effect of an imposed energy restriction on strength production measured with the Kin-Com was not limited by neural recruitment or inadequate energy production, but by the processes involved in excitation-contraction coupling. Therefore, the ultimate purpose of determining strength using the Kin-Com isokinetic dynamometer involved investigation of the effect of changes in muscle structure (as a result of the imposed energy restriction) on muscle strength. The Kin-Com measures strength in both a concentric and eccentric mode, which is useful in that both of these modes are used in running. The torque obtained from the concentric contractions was obtained from the upward phase of the lever arm, where the quadriceps muscle group shortened (the net muscle moment was in the same direction as the change in joint angle, and mechanical work was positive; Komi, 1986). The eccentric torque value was obtained from the downward phase of the lever arm (where the subjects were resisting the movement of the lever arm as it moved down) and involved a lengthening contraction of the quadriceps muscle group (net muscle moment was in the opposite direction to the change in joint angle, and mechanical work was negative; Komi, 1986). It is in the eccentric mode that the force and power capacities of the skeletal muscle are greatest (Komi, 1986). Also, to attain a certain force level requires much less motor unit activation in eccentric than concentric contraction (Komi, 1986). Additionally, oxygen consumption is much lower during eccentric exercise than in comparable concentric exercise (Komi, 1986). Peak and average torque value for each contraction type were used to describe muscle strength. Peak torque is the highest torque value obtained throughout the range of motion (65 degrees in this case), while average torque is the torque values averaged throughout the range of motion. Although both the peak and average torque values represent a measure of strength, peak torque attained in a single, high speed, velocity-  65  METHODS specific test is often used as a relative index of maximum power and strength (Komi, 1986). The functional capacity of the quadriceps muscles in the right leg were determined by a series of maximal knee extension and flexion movements with the subject sitting upright in the attached chair and the trunk stabilized by supporting straps. Muscle function was tested for both male and female subjects at the beginning, and on day 9 or 10 of the experimental testing period. Before every test session, the Kin-Com load cell was calibrated. To ensure gravitycompensated torque values for the data analysis, the right leg was weighed by the Kin-Com force transducer with the exercise arm positioned 15 degrees from the horizontal plane. This allowed the computer to adjust all subsequent force measurements for the calculated effect of gravity at each angle in the test range. The axis of the right knee was aligned with the axis of the Kin-Com exercise arm. Accuracy of this alignment was checked by allowing the subject to extend the leg while pushing against the shin pad which was positioned over the lower third of the leg. The peak and average torques generated for isotonic contractions and angular velocities of 30, 90,120, and 180 degrees/second were compared across all conditions. Velocities of 30 and 180 degrees/second were chosen specifically because these speeds are most often selected in other studies to measure peak torque as an indication of maximal strength, especially during the concentric phase of contraction (Alexander, 1990). This range of joint angular velocities also ensured a wide range of muscular contraction velocities and therefore a wide range of fiber recruitment The data collection form can be seen in Appendix 17. The subjects performed four consecutive extension and flexion movements with maximal intensity at each movement velocity with encouragement from the investigator. This was preceded by 4 practice submaximal extension-flexion movements at the specified movement velocity so that subjects were able to familiarize themselves with the equipment  66  _  _  METHODS  The order of movement velocities tested was randomized for each subject by having the subjects choose a movement velocity (written on a piece of paper) out of ajar. This ensured that there was no subject or investigator bias on the results of the test. The mean of the final three trials was used for statistical analysis because the first trial at each movement velocity has a variable torque value, as a result of the subject becoming accustomed to that movement velocity (Alexander, 1990). For the actual test, each subject was told to grasp the side of the table, lean against the backrest, and pull their right leg down against the calf pad as hard as possible, during the four flexion-extension cycles. During each test, frequent verbal encouragement was given. Concentric and eccentric torque scores were then compared to literature values. The concentric peak torques obtained at movement velocities of 30 and 180 degrees/second were given special attention as these indices are most often reported in other studies (Alexander, 1990). Frequently, torque values are divided by lean body mass, muscle mass, or fat-free mass (FFM) for the purpose of expressing strength values in relation to muscle mass. Muscle mass has been shown to be a determinant of muscle strength (Pronk et al, 1992). Therefore, in order to correct for muscle mass, the torque values were divided by the F F M .  67  ANALYSIS OF THE DATA  STATISTICAL ANALYSIS OF THE DATA  To determine if nutrition and exercise intervention in male subjects and to determine if nutrition intervention in female subjects had a significant impact on muscle protein degradation and the quadriceps muscle group function, the following statistical analyses were performed using BMDP 9D statistical program (BMDP Statistical Software, PC 90, Los Angeles, C A , 1994). For the male subjects, baseline and post-study parameters for the anthropometry, REE, RQ, calpain activity, and muscle function parameters were analyzed by performing the Hotelling t-squared statistical test. If there was a difference between the parameters, they were then compared with a correlated t-test. Similarly, the baseline and post-study parameters for the same variables as listed above were analyzed for the female subjects by performing the Hotelling t-squared statistical test. If there was a difference between parameters, they were then compared with a (2 x 2) (group x time) repeated measures A N O V A . Correlation analyses were performed between: substrate oxidation data and calpain activity: calpain activity and muscle function data. Correlations were calculated with the pre- and post-diet data, as well as the change in each parameter. Multiple stepwise regression was performed between the substrate oxidation data and calpain activity for pre- and post-diet data as well as the change in both variables. This was performed to determine: if calpain activity was determined by substrate oxidation parameters; and which substrate oxidation parameters were most important in determining calpain activity. Simple linear regression was performed on the muscle function data (dependent variables) and calpain activity (independent variable) for pre- and post-diet data as well as the change in both variables. This was done to determine: the extent to which muscle function was determined by calpain activity; which muscle function parameters were strongly determined by calpain activity. Simple linear regression was used to determine.  68  ANALYSIS OF THE  DATA  relationships between muscle function and calpain activity because there was one independent variable and several dependent variables. Comparisons were considered significant at a probability of p < 0.05. All results are presented as mean ± SEM.  69  SIGNIFICANCE  SIGNIFICANCE OF THE STUDY Because there is an increasing concern about body weight among Canadians, more information associated with the functional implications of a combined weight reduction and fitness program is required. Furthermore, knowledge regarding the effect of a low calorie intake in an athletic population, a practice which is commonly reported, may provide useful information for optimization of muscle performance. Thus, an energy restriction may have implications for performance capability of individuals (both sedentary and athletically trained) involved in various types of physical activity. Reports in the literature have indicated that an energy restriction results in an increase in fat oxidation. In turn, this was found to be associated with an increase in muscle proteolysis. A mechanism linking the above finding was speculated to include calpain because of the changes in the Z-line area of the myofibril (proteins in this area are substrates for calpain) occurring when fat oxidation is increased as a result of fasting and prolonged exercise. Disruption of sarcomere structure may result in a failure of force transmission, subsequently resulting in a decrease in muscle function. This study ascertained if there were links between substrate oxidation, calpain activity, and muscle function. Specifically, the relationship between substrate oxidation and calpain activity was assessed, to evaluate if substrate oxidation determined calpain activity. Also examined was the relationship between calpain activity and muscle function to evaluate if calpain activity determined muscle function.  HUMAN STUDIES This study received ethical approval from the University of British Columbia Clinical Screening Committee for Research and Other Studies Involving Human Subjects. See Appendix 18 for a copy of the acceptance form.  70  RESULTS  CHAPTER IV RESULTS 1.  SUBJECTS Eight men and fourteen women completed the study representing an attrition of  11% and 0% respectively. The ethnic profile of the male subjects who completed the study was Caucasian. The ethnic profile of the female subjects were Caucasian except one subject of mixed heritage (Chinese/Caucasian).  A. MALES Table 2 displays the male subject characteristics. All of the subjects were university students, and only one of the subjects was employed part-time. The types of activities that the males had previously participated in included: soccer (n=l), hockey (n=6), weight lifting (n=4), skiing (n=2), and running (n=l). The previous activities in which the male subjects were involved in are listed in Appendix 19. All of the males had been sedentary for the past two years as none of them were involved in more than 1 hour of activity per week. Their lack of activity was evident by their V O 2 max value (39.9 ± 2.9 ml/min/kg), which, for men, is not indicative of a trained person (Canadian Standardized Test of Fitness Operations Manual, 1986). The male subjects had body weights on average 20.0% higher than ideal body weight (refer to Appendix 20 for equation used for calculation of ideal body weight). The pre-study body weights ranged from 67.2 - 104.8 kg, of which 2 subjects were within the ideal body weight range for their height, and 6 subjects had weights which were larger than the ideal body weight for height. The mean Body Mass Index (BMI: 27.1 ± 1.4 kg/m^)  71  RESULTS was outside of the healthy limit of 18 to 25 kg/m (Hunt and Groff, 1990). The pre-study 2  BMI values ranged from 22.7 - 32.9 kg/m , where 2 of the subjects had BMI values 2  within healthy limits, and 6 had BMI values that were larger than 25 kg/m . 2  The percent body fat (19.9 ± 2.1%), which corresponded to a fat mass of 17.8 ± 3.7 kg, was within the desired subject criteria range of 10-20%. The pre-study percent body fat ranged from 12.9 - 28.2 % of which 4 subjects had body fat values that were outside of this range.  Table 2: Baseline characteristics of male subjects.  Variable  Pre-diet (n = 8)  Age (years)  22  ± I **  Height (cm)  175.9  ± 1.1  Weight (kg)  84.2  ± 4.9  Body Mass Index (kg/m )  27.1  ± 1.3  Triceps Skinfold (mm)  11.2  ± 3.5  Subscapular Skinfold (mm)  15.8  ± 3.7  Suprailiac Skinfold (mm)  18.1  ± 5.8  7.6  ± 1.8  2  Biceps Skinfold (mm) Sum of Skinfolds (mm)  59.2  ± 13.3  Body Fat (%)  19.9  ± 2.1  Total Body fat (kg)  17.8  ± 3.7  Fat Free Mass (kg)  68.5  ± 4.6  V02 Max (ml/min/kg)  39.9  ± 2.9  **Mean±Sl±M.  72  RESULTS  B. FEMALES Tables 3 displays the subject characteristics of all of the women as a group, and the women in each diet group at baseline. None of the female subjects were vegetarian. All of the female subjects had regular menstrual cycles and/or were taking oral contraceptives. Twelve of the female participants were university students. Six of the female subjects held part-time jobs, while one subject had the full time occupation of running her own business. One subject was unemployed throughout the duration of the study. Some of the subjects finished the study on day 9 of the study due to the timing of the measurements of the post-diet variables. The sample size on days 10 and 11 of the study was 6 and 3 respectively. The type of exercise the women participated in consisted of: running (n=14); biking (n=13); swimming (n=9); weight lifting (n=5); walking (n=2); field hockey (n=l); aerobics (n=3); downhill skiing (n=l); karate (n=l); lacrosse (n=l); rowing (n=2) (refer to Appendix 22 for a description of the activities in which the female subjects were involved). All of the women were involved in at least 3 different types of exercise throughout the duration of the study. All of the women had previously been involved in running, and had been exercising regularly and/or competitively for the last 9.4 ± 1 . 0 years. Evidence that these athletes were of the same caliber is shown by the V 0 2 max test results, where the 100% diet group ( V O 2 max = 49.4 ± 1 . 1 ml/min/kg) had a similar V O 2 max value to the 75% diet group ( V O 2 max = 48.5 ± 1 . 4 ml/min/kg) at the initiation of the study. Note that the average V O 2 max value of 49.0 ± 0.9 for all subjects is considered to be an excellent value for women between the ages of 20-29 years based on (Canadian Standardized Test of Fitness Operations Manual, 1987), and therefore indicated that the women were well-trained. Table 4 displays the baseline anthropometric measurements of the female subjects. Overall, the female subjects were well matched, as the two study groups did not differ  73  RESULTS significantly with respect to all of the characteristics listed in Table 3 or 4. The average body weight was within the desirable weight range (assuming a medium frame size; Metropolitan Height and Weight Tables, 1983). The pre-study body weights ranged from 51.2 - 75.6 kg, where 3 subjects had weights that were lower than their desirable weight, and 11 subjects had weights that were within the range of their desirable weight ranges. The female subjects had a mean BMI (22.1 ± 0.6 kg/m2)which was within the range set for subject inclusion. All female subjects except one, met the inclusion criteria of having a BMI within the healthy limits of 18 to 25 kg/m to participate in the study. The 2  pre-study BMI values ranged from 18.4 - 25.6 kg/m , where one subject had a BMI of 2  25.6 kg/m . Because this value closely approached the healthy BMI range, she was 2  accepted into the study. Table 3: Baseline characteristics of the female athletes according to the dietary subgroup.  Variable  100% diet  All (n = 14) 27 + 2  age (years)  (n = 7) 27 ± 2  7 5 % diet (n = 7) 27 + 4**  height (cm)  167.2  ± 1.6  164.5  ± 2.2  170  ± 1.7  weight (kg)  61.8  ± 1.9  58.5  ± 2.4  65.1  ± 2.4  BMI (kg/m )  22.1  ± 0.6  21.7  ± 0.9  22.6  +  V 0 2 max (ml/min/kg)  49.0  ± 0.9  49.4  ± 1.1  48.5  ± 1.4  years spent exercising  9.4  +  10.0  ± 1.9  8.9  2  1.0 .  **Mean±SEM. No significant differences between diet groups. Comparisons made by student's unpaired t-tests.  74  +  0.8  0.9  RESULTS  The average percent body fat for all of the subjects (20.0 ± 1 . 3 %), which corresponded to a fat mass of 12.6 ± 1 . 0 kg, fell within the desired subject criteria range of 16-26%. The pre-study percent body fat ranged from 12.6 - 26.9 %, of which 3 subjects had a percent body fat that was lower than 16% and 11 subjects had a percent body fat that was within the range of 16-26%. Table 4: Baseline anthropometric measurements of female athletes according to the dietary subgroup.  Variable  All, Pre-diet  100% Diet (n=7)  75% Diet (n=7)  (n=14) Triceps Skinfold (mm) Subscapular Skinfold (mm) Suprailiac Skinfold (mm) Abdominal Skinfold (mm) Axilla Skinfold (mm) Chest Skinfold (mm) Thigh Skinfold (mm) Sum of Skinfolds (mm) Body Density (kg/m3) Body Fat (%) Total Body Fat (kg)  13.7  ± 1.3  11.6 ± 1.7  16.1 ± 1.5**  14.7  ± 1.7  13.7 ± 2.7  15.8 ±  2.2  17.1  ± 2.1  114.2 ±  3.1  20.4 ±  2.1  18.0  ± 1.4  15.7 ±  2.0  20.6 ±  1.5  113.6  ± 1.6  12.4 ±  2.8  15.0 ±  1.5  6.2  ± 0.9  4.9 ±  1.1  7.8 ±  1.2  15.4  ± 1.1  14.2 ±  1.3  16.8 ±  2.0  97.2  ± 7.5  86.6 ±  12.6  107.8 ±  6.8  0.010  1.050  1.055  ± 0.003  1.060  ±  ± 0.002  20.0  ± 1.3  18.2 ±  2.2  21.8 ±  1.2  12.6  ± 1.0  10.9 ±  1.6  14.2 ±  1.1  47.7 ±  1.7  50.9 ±  1.8  49.3 ± 1.3 Fat-free Mass (kg) **Mean ± S E M . No significant differences between diet groups. Comparisons made by student's unpaired t-tests.  75  RESULTS  2. DIETARY  INTAKE:  A. MALES  Table 5 shows the males' mean nutrient intakes based on selected nutrients for the four days of diet records. The average energy intake per day (4321 ± 1160 kcal) supplied 175% of the calories recommended for a sedentary 22 year old male of height and weight of 176 cm and 84 kg respectively (averages for male subjects in this study; Food Processor JJ Nutrient Analysis Software, ESHA Research, Salem, OR). Dietary carbohydrate, protein and fat (as a percent of total energy) were respectively 60.7 ± 4.7%, 14.3 ± 2.4%, and 25.3 ± 2.9. Data was not obtained from one subject as he failed to return the four-day diet record. Of the 27 nutrients analyzed for which Recommended Nutrient Intakes (RNI) were available, the males exceeded the RNI for all of the nutrients which included: total vitamin A , thiamin, riboflavin, niacin, vitamin J36, vitamin B 1 2 , folacin, pantothenic acid, vitamin C, vitamin E, calcium, copper, iron, magnesium, potassium, phosphorus, sodium, selenium, and zinc. The subjects ingested at least double the RNI of the following nutrients: thiamin, riboflavin, niacin, vitamin B 1 2 , folacin, vitamin C, iron, potassium, phosphorus, sodium, and selenium. However, use of the Canadian database for assessing the micronutrient intake may be inappropriate due to the large percentage of missing values for these nutrients in the Canadian database (Barr et al, 1994). Therefore, assessment of micronutrient intake through the Canadian database should be approached with caution.  76  RESULTS  Table 5: Mean nutrient intakes of male subjects for four days*.  Daily Dietary Intakes  Mean ± SEM (n = 6) 4321 ± 116** 147.3 ± 33.2 665.7 ± 221.7 29.6 + 6.8 115.0 ± 21.63 39.8 ± 9.8 40.4 + 9.2 27.6 + 3.9 325.0 + 72.5 450.0 ± 187.4 687.7 ± 43.9 1137.7 ± 164.3 4.45 + 1.31 4.10 ± 1.19 45.1 ± 10.9 3.02 ± 0.37 7.20 ± 2.71 531 ± 141 8.27 ± 1.44 183 ± 82 15.77 ± 2.69 1841 ± 466 2.50 ± 0.58 28.3 ± 9.2 506 ± 104 4971 ± 598 2427 ± 600 6680 ± 1746 235.7 ± 75.4 18.93 ± 5.21 14.3 ± 2.4 60.7 + 4.7 25.3 ± 2.9 0.73 ± 0.23 1.30 ± 0.21 0.73 ± 0.09  Calories (kcal) Protein (g) Carbohydrates (g) Fiber (g) Total Fat (g) Saturated Fat (g) Monounsaturated Fat (g) Polyunsaturated Fat (g) Cholesterol (mg) Vitamin A - Carotene (RE) Vitamin A - Preformed (RE) Vitamin A - Total (RE) Thiamin (mg) Riboflavin (mg) Niacin (mg) Vitamin B6 (ug) Vitamin B 1 2 (ug) Folacin (ug) Pantothenic Acid (mg) Vitamin C (mg) Vitamin E (mg) Calcium (mg) Copper (mg) Iron (mg) Magnesium (mg) Potassium (mg) Phosphorus (mg) Sodium (mg) Selenium (ug) Zinc (mg) Protein (%) Carbohydrates (%) Fat (%) Polyunsaturated: saturated fat Sodium: Potassium Calcium :Phosphorus *Determined by Food Processor II Nutrient Analysis Software (ESHA Research, Salem, OR) prior to the study. **Mean±SEM. v  77  RESULTS  B.  FEMALES  Table 6 shows the females' mean nutrient intakes based on selected nutrients for the four day diet records. The average caloric intake per day was 2052 ± 1 5 3 kcal which is 17% lower than that recommended for a very active 27 year old female of height and weight of 167 cm and 62 kg respectively (averages found for females in this study; Food Processor U Nutrient Analysis Software, E S H A Research, Salem, OR). The 100% diet group satisfied 77 ± 10% of the caloric requirement, while the 75% group satisfied 90 ± 7% of the caloric requirement at baseline. Dietary carbohydrate, protein, and fat (as a percent of total energy) were respectively 54.6 ± 3.7 %, 14.6 ± 1.1%, and 30.8 ± 4.2% respectively. The two diet groups ingested similar proportions of macronutrients in their diets. The only significant difference for the nutrient intakes between the 75% and 100% diet group was found with total vitamin A intake, where the 100% diet group had a larger total vitamin A intake than the 75% diet group (p < 0.01). Of the 27 nutrients analyzed for which Recommended Nutrient Intakes (RNI) were available, the female subjects as a whole exceeded the RNI for 23 of the nutrients: total vitamin A , thiamin, riboflavin, niacin, vitamin B-6, vitamin Bi2, folacin, vitamin C, vitamin E, calcium, copper, iron, magnesium, phosphorus, sodium, potassium, selenium, and zinc. They had lower than recommended intakes for pantothenic acid and copper. Of these nutrients, the 100% diet group had lower intakes for all of these nutrients, while the 75% diet group had lower than recommended intakes for all nutrients in addition to the monounsaturated fat intake. Both diet groups ingested more than the recommended intakes by a large margin for vitamin C , potassium, and sodium. As mentioned before, assessment of micronutrient intake through the Canadian database should be approached with caution.  78  RESULTS  Table 6: Mean nutrient intakes of the female athletes for four days, according to the dietary subgroup.*  Daily Dietary Intakes  All Subjects (n = 14)  100% Diet Group (n = 7)  75% Diet Group (n = 7)  Calories (kcal)  2052  ± 153  1928  ± 253  2209  ± 140  Protein (g)  72.2  ± 5.4  68.8  ± 7.7  76.4  ± 16.2  314.2  ± 18.50  Carbohydrates (g)  284.2  ± 19.76  260.2  ± 29.6  Fiber (g)  23.7  ± 2.5  21.0  ± 2.5  27.0  ± 4.6  Total Fat (g)  73.5  ± 10.4  70.3  ± 17.8  77.5  ± 10.4  Saturated Fat (g)  25.5  ± 4.2  23.9  +  27.5  ± 4.7  Monounsaturated Fat (g) Polyunsaturated Fat (g) Cholesterol (mg)  28.0  ± 4.1  29.0  ± 6.9  26.8  ± 4.8  18.2  ± 2.1  15.6  ± 3.4  21.5  +  17.8  4.7  196.3  ± 45.92  221.4  ± 78.9  165.0  ± 40.9  516.8  ± 121.8  630.7  ± 201.8  374.3  ± 94.3  387.6  ± 56.1  390.6  ± 75.9  383.8  ± 96.3  904.1  ± 92.3  +  757.8  ± 6.0  Thiamin (mg)  1.68  ± 0.14  1.53  ± 0.22  1.87  ± 0.15  Riboflavin (mg)  1.79  ± 0.11  1.70  +  1.90  ± 0.13  Niacin (mg)  18.6  ± 1.8  19.0  ± 2.6  18.2  +  Vitamin B6 (ug)  1.74  +  0.26  1.78  ± 0.30  1.68  ± 0.49  Vitamin B12 (ug)  2.82  ± 0.52  3.11  ± 0.83  2.45  ± 0.62  Vitamin A - Carotene (RE) Vitamin A - Preformed (RE) Vitamin A - Total (RE)  Folacin (ug) Pantothenic Acid (mg)  296 4.78  Vitamin C (mg)  161  Vitamin E (mg)  11.92  Calcium (mg)  897  Copper (mg)  1.86  ± 38 ± 0.47 ± 28 ± 1.53 ± 64 ± 0.21  a  1021.2  259 4.52 174  151.5  0.16  ± 48 ± 0.43 ± 36  342 5.10 144  +  2.8  59  ± 0.97 ± 50 ± 2.03  9.84  ± 1.86  14.53  810  ± 100  1004  ± 33  1.54  ± 0.20  2.27  ± 0.32  RESULTS 16.1  ± 1.6  14.2  Magnesium (mg)  348  ± 42  302  Phosphorus (mg)  1326  ± 127  1188  ± 132  1498 + 223  Sodium (mg)  2240  ± 273  2330  ± 454  2127  ± 312  Potassium (mg)  3050  ± 340  2840  ± 421  3313  ± 598  121.0  ± 13.7  106.3  ± 14.6  139.3  ± 23.9  Zinc (mg)  9.55  ± 0.77  8.83 + 1.08  10.45  ± 1.00  Protein (%)  14.2  ± 0.7  14.6  ± 1.1  13.8  ± 1.1  Carbohydrates (%T  55.2  ± 2.2  54.6  ± 3.7  56.0  ± 2.5  Fat (%)  30.8  ± 2.5  30.8  ± 4.2  30.8  ± 4.7  Polyunsaturated: saturated fat Sodium:Potassium  0.73  ± 0.13  0.74  ± 0.12  0.73  ± 0.27  0.85  ± 0.15  0.92  ± 0.21  0.75 + 0.21  Calcium:Phosphorus  0.69 + 0.04  0.66  ± 0.02  0.73  Iron (mg)  Selenium (ug)  ± 1.94  18.5  ± 2.5  ± 44  404  ± 74  ± 0.09  *Determined by Food Processor II Nutrient Analysis Software (ESHA Research, Salem, OR) prior to the study. **Mean ± S E M . S i g n i f i c a n t difference between 75% and 100% diet group, p < 0.01. Comparison's made by student's unpaired t-test.  80  RESULTS  3.  ANTHROPOMETRY  A. MALES Anthropometric measures of male subjects pre and post-treatment are shown in Table 7. The total amount of weight lost within the experimental diet period was 1.5 ± 0.3 kg (p < 0.01). Thus, the percent weight lost was 1.7 ± 0.3 %. Also, BMI decreased by 0.5 ±0.1 (p < 0.01) as a result of the weight change. Although skinfold measures at each specific site did not change independently, the sum of the four skinfolds resulted in a 5.2 ±1.9 mm decrease (p < 0.05) which corresponded to a decrease in body fat (-1.4 ± 0.2 kg, p < 0.01; -1.3 ± 0.4%, p < 0.05).  81  RESULTS  Table 7: Anthropometric measures of male subjects before and after the dietary and exercise treatment.  Variable  Pre-treatment (n = 8)  Post-treatment (n = 8)  Change  Weight (kg)  84.2  ± 4.9  82.7  ± 4.6  1.5  Body Mass Index  27.1  ± 1.3  26.6  ± 1.3  -0.5  ± o.ia  Triceps Skinfold (mm)  11.2  ± 3.5  10.0  ± 2.2  -1.2  ± 2.3  Subscapular Skinfold (mm) Suprailiac Skinfold (mm) Biceps Skinfold (mm)  15.8  ± 3.7  14.6  ± 2.4  -1.2  ± 3.6  18.1  ± 5.8  21.1  + 8.3  3.0 ± 4.4  7.6  + 1.8  7.8  + 2.3  0.2  Sum of Skinfolds (mm) Body Fat (%)  59.2  ± 13.3  54.0  ± 12.9  -5.2  ± 1.9b  19.9  ± 2.7  18.7  ± 2.9  -1.3  ± 0.4b  Total Body fat (kg) (TBF) Fat-free mass (kg) (FFM)  17.8  + 3.7  16.5  ± 3.7  -1.4  ± o.2  66.4  ± 3.9  66.2  ± 3.1  -0.1  ± 0.6  (kg/m )  ± 1.3**,a  2  **Mean±SEM. Significant difference between pre- and post-treatment, p < 0.01. Significant difference between pre- and post-treatment, p < 0.05. Comparisons made with correlated t-tests. a  D  82  ±  1.8  a  RESULTS B. F E M A L E S Anthropometric measures of female subjects, pre- and post-diet, are shown in Table 8. The body weight in the subjects of the 100% diet group did not change, while body weight decreased (-1.7 + 0.3 kg; p < 0.001) in the subjects of the 75% diet group. This corresponded to a 2.6 ± 0.4% weight loss in the 75% diet group. Furthermore, BMI decreased by 0.7 ± 0.03 kg/m (p < 0.01) in the 75% diet group as a result of the weight 2  change. There was a decrease in the sum of skinfolds (-15.7 ± 2.4 mm; p < 0.01) in the 75% diet group which corresponded to a decrease in body fat (-2.3 ± 0.4 kg, p < 0.01; -2.9 ± 0.6 %, p < 0.05). These changes were due to decreases in the following skinfold measures (p < 0.05): triceps, suprailiac, axilla, and chest. No such differences in body fat, sum of skinfolds, or individual skinfold measures were found for subjects in the 100% diet group.  83  RESULTS Table 8: Anthropometric measures of female athletes according to the dietary subgroup before and after the respective diet period.  Variable Pre-diet  100% Diet (n = 7) Post-diet Change  Pre-diet  7 5 % Diet (n = 7) Post-diet Change  Weight (kg) 58.5 ± 2.4  58.5 ± 2.4  -0.04 ± 0.03  65.1 ± 2.4  63.4 ± 2.2  Body Mass Index (kg/m ) Triceps Skinfold (mm) Subscap-uiar Skin-fold (mm) Suprailiac Skinfold (mm) Abdominal Skinfold (mm) Axilla Skinfold (mm) Chest Skinfold (mm) Thigh Skinfold (mm) Sum of Skinfold (mm)  21.7 ± 0.9  21.7 ± 0.9  -0.04 ± 0.03  22.6 ± 0.8  21.9±0.8  11.6+ 1.7  13.2+ 1.7  1.6 ± 0.7  16.1 ± 1.5  14.8 ± 0.8  -1.1 ± 0 . 9  13.7 ± 2.7  13.6 ± 2 . 6  -0.1 ± 0.6  15.8 ± 2.2  13.4 ± 1.6  -1.6 ± 2 . 2  14.2 ± 3.1  14.0 ± 2.5  -0.1 ± 1.0  20.4 ± 2 . 1  14.6 ± 2.3^  -4.4 ± 1.4C  15.7 ± 2.0  16.3 ± 2.3  0.6 ± 1.2  20.6 ± 1.5  16.1 ± 0.6  -4.8 ± 2.2C  12.4 +2.8  12.4 ± 2.3  -0.3 ± 1.0  15.0 ± 1.5  12.3 ± 1.2a  -2.1 ± 0.7  4.9 ± 1.1  4.3 ± 0.4  -0.6 ± 1.2  7.8 ± 1.2  4.8 ± 0.5  -3.0 ± 0.9  14.2 ± 1.3  14.4 ± 1.0  0.3 ± 1.2  16.8 ± 2.0  16.1 ± 1  86.6 ± 12.6  88.2 ± 11.2  1.6 ± 3.9  107.8 ± 6.8  92.1 ± 4 . 8  1.060 ± 0.010  1.060 ± 0.004  0.0001 ± 0.001  1.050 ± 0.002  1.060 ± 0.002d  18.2 ± 2.2  18.1 ± 1.9  -0.1 ± 0.8  21.8 ± 1.2  18.9 ± 0.9^ -2.9± 0.6C  10.9 ± 1.6  10.7 ± 1.4  -0.2 ± 0.5  14.2 ± 1.1  12.0 ± 0.9  Fat-Free 47.7 ± 1.6 0.05 ± 0.5 50.9 ± 1.8 47.7 + 1.7 Mass (kg) Mean ± S E M . significant difference within group, pre- versus post-diet, p<0.05-  51.3 ± 1.6  a  -1.7 ± 0.3**. b -0.7 ± 0.03b  a  2  Body Density (kg/m3) Body Fat (%) Total Body Fat (kg)  a  84  a  C  -0.8 ± 1.3  d  -15.7 ± 2.4e 0.007 ± 0.001*  d  -2.3 ± 0.4e  0.5 ± 0.5  RESULTS  bsignificant difference between the change in the 100% group versus the change in the 75% group, p < 0.001. Significant difference between the change in the 100% group versus the change in the 75% group, p < 0.05. "significant difference within group, pre- versus post-diet, p<0.01. Significant difference between the change in the 100% group versus the change in the 75% group, p < 0.01. Comparisons made by a 2 x 2 R M A N O V A for each variable.  85  RESULTS  4.  EXERCISE  DATA:  A. MALES Subjects exercised (ie jogging) for a 30 minute session each day throughout the study period. The average energy expended per session is shown in Table 9. The highest energy expended occurred on day 1 of the study (314 + 27 kcal/day) while the lowest occurred on day 8 of the study (253 ± 1 1 kcal/day). Exercise data for one subject was not obtained as he failed to record his heart rate values. In order to evaluate whether or not there was a decrease in energy expenditure with the consecutive days of exercise a trend analysis was performed. No linear, quadratic, or cubic trend was significant for this set of data. The energy expended was not significantly different between any of the days exercised.  86  RESULTS  Table 9: Average energy expended per exercise session for male subjects*. Day 1  Energy Expenditure (Kcal/30 min exercise session) (n = 7) 314 ± 2 7 * *  2  306 + 20  3  299 ± 2 1  4  274 ± 29  5  296 ± 23  6  292 ± 18  7  290 ± 29  8  253 ± 11  9  270 ± 16  *Determined from heart rate values, (which was then used to determine energy expenditure through the calculation of oxygen consumption from heart rate; refer to Appendix 17 for equation relating heart rate and oxygen consumption). Mean ± S E M .  RESULTS  Heart rate values determined at specific time points during the exercise session are shown in Table 10. The average maximum heart rate for all of the subjects was 186 ± 7 beats/minute. Heart rate reserve follows a trend of increasing throughout the 30 minute exercise session. Heart rate and heart rate reserve increased linearly withtimein a significant manner, (p < 0.0001, p < 0.0000 respectively) determined by trend analysis. The highest heart rate reserve occurred between 15 and 25 minutes of exercising. Table 10: Average exercise heart rate and reserve heart rate values for male subjects at various time points during the 30 minute exercise session^.  Time (minutes)  Heart Rate (beats/min)  Heart Rate Reserve*  (n = 7)  (n = 7)  0  82 ± 3  45 ± 3**  5  146 ± 5  79 ± 3  10  150 + 5  81 ± 2  15  152 ± 6  82 ± 3  20  152 ± 6  82 ± 3  25  151 ± 6  82 ± 3  30  148 ± 5  80 ± 3  §Average values for a nine day period. * Percent maximum heart rate. **Mean ± S E M .  88  RESULTS  Throughout the study, the average energy expenditure from exercise for each subject was 288 ± 1 8 kcal/day. This corresponded to an energy deficit of 12 ± 0.1 % when energy expenditure was expressed relative to the average maintenance energy intake (2491 ± 2 1 kcal; refer to Table 11). This was similar to the desired energy deficit of 11%. The actual dietary energy deficit created was 22 ± 0.5% (548 ± 5 kcal), which (when added to the deficit created by exercise) gave rise to a total deficit of 34 ± 1 % (841 ± 26 kcal).  89  RESULTS  Table 11: Energy intake and energy expenditure of the male subjects.  Variable  Value (n = 8)  (A) Maintenance energy requirement (kcal)  2491 ±  (B) Energy expenditure from  21**  288 ± 18  exercise*(kcal/day) (C) Energy deficit from diet (kcal/day)  548 ± 5  (D) Total deficit (kcal/day) (B + C)  841 + 26  (E) Net Caloric Intake (kcal/day) ( A - D )  1647 ± 13  (F) % Energy Deficit of Maintenance Energy  12 ± 1  from Exercise (B/A x 100) (G) % Energy deficit of Maintenance energy  22 ± 1  from diet (C/Ax 100) (H) % Total Energy deficit of Maintenance  34+1  Energy (D/A x 100) % Energy Requirement Satisfied  66 ± 1  (E/AxlOO) **Mean + S E M . *Determined from the relationship between heart rate and oxygen consumption.  90  RESULTS  B. FEMALES  Table 12 presents the activity characteristics of the female athletes. Although the distance run per week (37 ± 4 km) was not greater than or equal to 50 km per week (one of the inclusion criteria; refer to Appendix 22 for the distance run per week by each subject), all of the subjects were involved in at least one other aerobic activity other than running, as was evident by the large amount of time spent exercising each day (see Appendix 21 for activities in which the female subjects were involved and Appendix 23 for the time spent by each subject exercising each day). The activity characteristics did not differ between the two diet groups.  Table 12: Activity characteristics of female athletes according to dietary subgroup during the study.  1 0 0 % Diet (n = 7 )  7 5 % Diet (n = 7 )  Run distance per week 37.1 ± 3.7 (km/week)  42.4 ± 4.3  31.9 ± 5.6**  Time spent exercising each day (min/day)*  99.9 ± 6.4  105.2 ± 10.1  94.7 ± 8.0  Energy Expenditure from exercise Qccal/day)§  832 ± 97  829 ± 119  834 ± 142  Characteristic  All (n = 14)  **Mean ± S E M . No significant differences. Comparisons made by student's unpaired t-tests. •Calculated from daily exercise recording forms. §Calculated by taking average of days 1 to 9; determined by the Weight Loss Programmer (Version 3.1, Ohio Distinctive Software, Cleveland, Ohio, 1992).  91  RESULTS  Table 13 displays the energy expended during exercise per day for the female subject group over a nine day period. The 100% and 75% diet groups did not differ with respect to the amount of energy expended per day on any day during the study except on day 6 where the energy expenditure for the 100% diet group (1009 ± 128 kcal/day) was significantly larger than the, 75% diet group (673 ± 66 kcal/day). Appendix 25 contains the results of energy expended per day for the female subjects over 11 days. The energy expended by the female subjects on days 10 and 11 was not included in Table 13 because the sample size was too small to represent values obtained for the entire group. The average exercise energy expenditure of days one to nine for the entire group of subjects, the 100% diet group, and the 75% diet group were respectively: 833 ± 97 kcal/day, 829 ± 1 1 9 kcal/day, and 834 ± 142 kcal/day. The average exercise energy expenditure on days one to nine did not differ between groups. The highest energy expended per day for the entire group of subjects, and subjects in the 75% diet group occurred on day nine while the highest energy expended for the 100% diet group occurred on day 6. The lowest energy expended per day for the entire group of subjects, the 100% diet group, and the 75 % diet group occurred on days 7, 7, and 6 respectively. The slopes of the graphs of days exercised versus energy expended for the diet groups were compared to determine: if the energy expenditure progressively decreased with the amount of days exercised, and if this differed between groups. The resulting slope values for the entire group of subjects, the 100% diet group, and the 75% diet group when considering days 1 to 9 were respectively: -7.80 ± 8.36 kcal/days , -12.07 ± 10.5 2  kcal/days , and -3.53 ± 13.66 kcal/days (Figure 1). The slope values were not 2  2  significantly different between the two diet groups and no significant linear trends were found within each diet group with successive days of exercise.  92  Figure 2: Energy expenditurefromexercise throughout the diet period.  A l l subjects 1000  100% Diet Group 75% Diet Group  800  1  600 4  6  Time (days)  93  10  RESULTS  Table 13: Average energy expended from exercise per day for female athletes according to the dietary subgroup.  Day 1  Sample size (All A l l subjects) 14 923 ± 97  Energy Expenditure (kcal/day)* 100% Diet group 75% Diet group 914 ± 156 (n = 7)  932 ± 129 (n = 7)**  2  14  796 ± 95  751 ± 108 (n = 7)  841 ± 164 (n = 7)  3  13  789 ± 73  784 ± 88 (n = 7)  794 ± 124 (n = 7)  4  13  862 ± 97  943 ± 108(n = 7)  780 ± 164 ( n = 7)  5  13  8 8 1 ± 110  837 ± 108 (n = 5)  920 ± 121 (n = 7)  6  13  854 ± 87  1009 ± 128 (n = 7)  673 ± 66 (n = 6)  7  11  729 ± 90  710 ± 171 (n = 5)  744 ± 102 (n = 6)  8  14  741 ± 7 6  7 1 7 ± 9 8 ( n = 7)  765 ± 125 (n = 7)  9  13  926 ± 150  795 ± 110 (n = 7)  1056 ± 283 (n = 6)  a  •Determined by the Weight Loss Programmer (Version 3.1, Ohio Distinctive Software, Cleveland, Ohio, 1992). **Mean±SEM. Significant difference between 100% and 75% diet groups, p < 0.05. Comparisons made by student's unpaired t-tests. a  94  RESULTS  5. SUBSTRATE  OXIDATION:  A. MALES Table 14 displays the resting energy expenditure data for the male subjects. Their were no changes in the resting energy expenditure and the resting R Q value as a result of the diet and exercise treatment. However, it should be noted that data for only three subjects were used to make these comparisons.  Table 14: Resting energy expenditure data during diet treatment* for male subjects. Variable REE§ (kcal/day)  Pre-treatment (n = 3)  Post-treatment (n = 3)*  1860+ 155  1920±122**  (kcal/kg body 21.6 ± 0.9 weight) (kcal/kg fat27.4 ± 0 . 1 free mass) R Q (VCO2/VO2) 0.84 ± 0.03  22.8 ± 1.0 28.4 ± 0.7 0.80 ± 0.09  Measured at the beginning and end of each diet treatment. $Data for 3 subjects was not assessable; 2 subjects could not schedule the tests. § Calculated by formula of Weir (1949). **Mean±SEM. No significant differences. Comparisons made by correlated t-tests.  95  RESULTS  Post-prandial R Q values of male subjects pre- and post-treatment are presented in Table 15. The post-prandial R Q values significantly decreased (p < 0.05) at the following time points: 60 minutes (-0.09 ± 0.01); 120 minutes (-0.04 ± 0.01); 150 minutes (-0.08 ± 0.01). The decrease in the post-prandial RQ at 90 minutes(-0.02 ± 0.01) almost showed a significant difference (p = 0.07). Table 15: Post-prandial R Q * values after consumption of a test breakfast for male subjects.  Time (minutes)  Pre-treatment* (n =3)  Post-treatment (n = 3)  30  0.90 ± 0.02  0.83 ± 0.00**  60  0.89 ± 0.02  0.80 ± 0.02^  90  0.86 ± 0 . 0 1  0.83 ± 0.02  120  0.86 ± 0.03  0.83 ± 0.03a  150 0.88 ± 0.02 *Measured at the beginning and end of the diet treatment. **Mean ± S E M . Significant difference, p < 0.05. Comparison made by correlated t-tests. a  96  0.80 ± O.Oia  RESULTS  B.  FEMALES  The resting energy expenditure data of the female subjects pre- and post-diet is shown in Table 16. The resting energy expenditure did not differ between the diet groups at each time point, or pre- and post-diet within each diet group, even when corrected for body weight and F F M . The resting RQ value of the 75% diet group decreased (-0.06 ± 0.02; p < 0.05), while the value for the 100% diet group did not change.  Table 16: Resting energy expenditure data during diet treatment* for female athletes according to the dietary subgroup.  l Parameter  1 0 0 % Diet (n = 7)  7 5 % Diet (n = 7)  Pre-diet  Post-diet  Pre-diet  Post-diet  (kcal/day)  1344 ± 7 8  1341 ± 4 6  1511 ± 9 8  1464 ± 6 3 * *  (kcal/kg body  22.9 ± 0.7  23.2 ± 0.5  23.2 ± 1.0  23.1 ± 0.8  28.1 ± 1.1  28.4 ± 0.8  29.7 ± 1.7  28.6 ± 1.0  REE§  weight) (kcal/kg FFM)  RQ (VCO2/VO2) 0.87 ± 0 . 0 3 0.84 ± 0.02 0.83 ± 0.03 0.77 ± 0.02 >P * Measured at the beginning and end of each diet period § Calculated by the formula of Weir (1949) **Mean ± S E M . significant difference within the diet group, pre- versus post-diet (p<0.05). b significant difference between the change in the 100% diet group versus the change in the 75% diet group, (p<0.05). Comparisons made with a 2 x 2 R M A N O V A for each variable. a  a  97  RESULTS Table 17 displays the post-prandial RQ values pre- and post-diet in the female athletes according to the dietary subgroup. There were no significant differences in t he post-prandial R Q values between the 100% and 75% diet groups either pre-diet or postdiet. The post-prandial RQ values for the 75% group decreased from the pre-diet value at the following time points: 30 minutes (-0.07 ± 0.03; p < 0.05); 120 minutes (-0.10 ± 0.02; p < 0.001); 150 minutes (-0.09 ± 0.03; p < 0.05). The changes in the post-prandial R Q values for the 75 % group were significantly larger than changes occurring in the 100% diet group at 120 minutes (p < 0.001) and 150 minutes (p < 0.05). There was no change in the post-prandial R Q values for the 100% diet group, pre- and post-diet.  98  .2  00  oo ©  8| o  CN  o ©  CL  +-»  o  ^  ©'  ©  ©  +1  CN  © o  OO 00  CN ©  ©  ©  o o © o V X 3 3  +1  +1 O  CN  8-1©  ©  C/J  o  00  +1 ON 00  ON  o\ ©  r©  CS  o ©  a , a, 3  3 oo  ob ©fl  o T-l  TJ  +i *  -3  +1 oo  +1 CO  oo  o  o  co  .2  •a T J  co o o  co co  CO  Si B  .2  +1 r-  oo  +1 o  o.  -3 & cB  +1  CN  eo i>  CN 00  o ©  ©  •8-8 a> jo  +1 CO  00  o  o  o © © o S3 V v  CN  o o  • 1-12^ H->  .a  +1 o CN o o o  +1  T3  O  'G  00  eo  CO «0 T3 T J  to 2S  °  eo  +1  +1  cn o  ©  ON 00  co o  ©  ©  CO <H-I  o •a a  ^1 T+l -1  eo +1 « •o 00 CN OO o »fl  CN  Sj o oo  o  ©  ©  OH OH  •o  I TJ  o  OH CO  co  1 | .3 a  !>& oPI § OH OH  ^ 3  l-l  <-l  CO  eo  6fl  6fl  .S  00  S  §§  • ^H  I—I  2  1^  Sb » «  2 •a  OH OH  •*-»  o  CO  OH OH  •S .5  CN X CN  &  CO  +1 o © ON  +1 in  ON  ©  ©  X3 CN ©  ©  TJ  OH S3 O ©fl  .S3 co 5d *H 1=0 -a -o =3 s  l-M •> •)_! H)-J C^C C  +1  ^  CO  o o  co ,co £  co •-6it* o r-  99  d  c  'Slijs i -«^ so ;r1  GO «3  MI*  • ,  o  U  RESULTS  In order to evaluate whether the change in RQ over time was significantly different pre- and post-diet within each diet group, and at each time point between each diet group, the slopes of the lines of time versus RQ were evaluated statistically. No significant differences were found between slope values pre- and post-diet within each diet group, and at each time point between each diet group.  6. MUSCLE  FIBER  ANALYSES:  A. MALES There was no significant difference in calpain activity pre- (9.40 ± 2.30 units/g wet weight) and post-treatment (13.63 ± 2.59 units/g wet weight) where the change in activity was 4.23 ± 4.60 units/g wet weight (n = 4) as assessed by correlated t-tests.  B. FEMALES  Table 18 shows the pre- and post-diet myofibrillar calpain activity for the female subjects. There were no differences in myofibrillar calpain activity between the diet groups at either time point. Furthermore, there were no differences in pre- and post-diet myofibrillar calpain activity within each diet group.  Table 18: Myofibrillar calpain activities of female athletes according to the dietary subgroup before and after dietary period.  Diet Type Pre-Diet  Calpain Activity (units/g wet weight) Post-Diet Change  100%* (n = 6)  28.82 ± 4.9  34.84 ± 10.57  6.02 ± 7.43**  75%* (n = 6)  20.82 + 2.49  42.98 ± 12.49  22.17 ± 14.34  **Mean + S E M . No significant differences. Comparisons made with a 2 x 2 R M A N O V A .  100  RESULTS  7.  MUSCLE  A.  FUNCTION:  MALES  Table 19 shows the torque values for the eccentric contractions of the quadriceps muscles for the male subjects. There were no significant differences between pre- and post-diet values for any of the average or peak torque values for eccentric contractions. Note that the sample size for the average torque parameters was 6 instead of 8 because pretreatment data was inaccessible for 2 subjects because baseline muscle function measurements were obtained from a previous study which did not look at the average torques.  Table 19: Eccentric contractions of quadriceps muscles of male subjects before and after the diet and exercise period. Velocity  Pre-treatment  Post-treatment  Change in Torque  (degrees/sec) Torque (Nm) Peak torque (n = 8)  Torque ( Nm)  (Nm)  30  179 ± 1 9  177 ± 1 3  -2 ± 1 8 * *  90  177 ± 18  206 ± 7  28 ± 2 1  120  177 ± 2 1  195 ± 1 9  18 ± 2 7  180  175 ± 2 5  175 ± 2 0  0±25  Average torque (n = 6) 30  173 ± 9  153 ± 1 1  -20 ± 1 1  90  158 ± 1 0  156 ± 1 4  -2 ± 2 2  120  140 ± 1 2  158 ± 1 6  18 ± 2 5  180  146 ± 1 5  137 ± 18  - 9 ± 16  **Mean±SEM. No significant differences. Comparisons made with correlated t-tests.  101  RESULTS Table 20 shows the torque values for the concentric contractions of the quadriceps muscles for the male subjects. There were no significant differences between pre- and post-diet values for any of the concentric peak torque values. However, the concentric average torque obtained at a movement velocity of 30 deg/sec decreased (-22 ± 6 Nm; p < 0.05) from the pre-diet value.  Table 20: Concentric contractions of quadriceps muscles of male subjects before and after diet and exercise treatment. Velocity  Pre-treatment  Post-treatment  Change in Torque  (degrees/sec) Torque (Nm) Peak torque (n = 8)  Torque (Nm)  (Nm)  30  163 ± 24  157 ± 10  - 5 ± 16**  90  150 ± 14  159 ± 18  9 ± 18  120  154 ± 20  165 ± 17  11 ± 1 0  180  144 ± 16  152 ± 16  8±8  Average torque (n = 6) 30  144 ± 18  123 ± 12  -22 ± 6  90  132 ± 16  133 + 21  0 ± 11  120  138 + 21  142 ± 23  4±6  180  125 ± 16  130 ± 20  5±9  **Mean±SEM. Significant difference between pre- and post-diet values, p < 0.05. Comparisons made with correlated t-tests. a  102  a  RESULTS  In order to evaluate if the change in torque with increasing movement velocity was significantly different pre- and post-treatment, the slope of the graph of movement velocity versus torque value was evaluated statistically. There were no significant differences between any of the pre- and post-treatment slope values for all contraction and torque types.  103  RESULTS  B. FEMALES  The peak and average eccentric torques of the female subjects according to the dietary subgroup pre- and post-diet are displayed in Table 21. The eccentric average torque of the 100% diet group increased at a movement velocity of 90 degrees/second (36 ± 19 N m ; p < 0.05), while the eccentric average torque of the 75% diet group at a movement velocity of 180 degrees/second increased (19 ± 13 N m ; p < 0.05). There were no differences in the eccentric peak torque within each diet group or between the diet groups.  Table 21: Eccentric contractions of quadriceps muscles of female athletes according to the dietary subgroup before and after the diet period.  Velocity  100 % Diet (n = 7)  7 5 % Diet (n = 7)  (deg/sec)  (Nm)  (Nm)  pre-diet  post-diet  pre-diet  post-diet  30  111± 17  121 ± 1 6  121 ± 11  127 ± 1 3 * *  90  86 ± 15  125 ± 19  112 ± 9  124 ± 1 2  120  129 ± 16  131 ± 1 9  121 ± 1 0  126 ± 15  180  132 ± 18  138 ± 17  116 ± 13  135 ± 15  30  90 ± 14  100 ± 14  100 ± 9  115 ± 13  90  69 ± 15  98 ± 1 5  91 ± 7  101± 10  120  97 ± 13  9 4 ± 15  98 ± 6  107 ± 1 1  180  95 ± 14  97 ± 14  85 ± 12  106 ± 9  Peak torque  Average torque a  **Mean ± S E M . Significant difference from pre-diet value, p < 0.05. Comparisons made with a 2 x 2 R M A N O V A for each condition. a  104  a  RESULTS Table 22 shows the peak and average concentric torques of the female subjects according to the dietary subgroup before and after the diet period. The concentric peak torque in the 75% diet group changed from the pre-diet value (-10 ± 6 Nm; p < 0.05). Furthermore, this change was significantly larger than that seen in the 100% diet group (p < 0.05). The concentric average torque at a movement velocity of 30 degrees/second in the 100% diet group increased (11 ± 5 Nm; p < 0.05).  Table 22: Concentric contractions of quadriceps muscles of female athletes according to the dietary subgroup before and after the diet period.  Velocity (deg/sec)  100 % Diet (n = 7) (Nm) pre-diet post-diet  75% Diet (n (Nm) pre-diet  7)  post-diet  Peak torque 30  118 ± 11  124 ± 8  116 ± 8  109 + 9 **  90  107 ± 15  116 ± 6  108 ± 10  97 + 11  120  106 ± 10  114 ± 6  104 ± 8  93 ± 9 >  180  102±9  106 ± 9  98 ± 8  101 ± 5  30  98 ± 9  110 ± 6 >  96 ± 6  93 ± 7  90  94 ± 13  102 ± 6  96 ± 7  85 ± 10  120  92 ± 10  97 ± 7  92 ± 7  84 ± 8  180  85 ± 13  88 ± 8  86 ± 6  83 ± 6  a  b  Average torque a  b  Mean ± S E M . Significant difference from pre-diet value, p < 0.05. Significant difference between the change in the 100% diet group versus the change in the 75% diet group, p < 0.05. Comparisons made with a 2 x 2 R M A N O V A for each condition. a  b  105  RESULTS  To evaluate whether the change in torque with increasing movement velocity was significantly different pre- and post-diet within each diet group, and at each time point between the 100% and 75% diet groups, the slope of the graph of movement velocity versus torque value was evaluated statistically. There were no significant differences between any of the pre- and post-diet slope values within each diet group for any contraction or torque type. There were also no significant differences between the 100% and 75% diet group slope values at each time point.  106  RESULTS 8.  FACTORS RELATING TO CHANGES IN CALPAIN ACTIVITY  A. MALES Stepwise multiple regression analysis was not performed on the calpain data with substrate oxidation parameters incorporated as the subject number was too low to provide representative results. i  B. FEMALES Stepwise multiple regression analysis was performed on the calpain data incorporating substrate oxidation variables (REE, post-prandial RQ values at all time points) to determine which substrate oxidation variables were most important in determining calpain activity. The pre-diet substrate oxidation parameters did not predict the pre-diet calpain activity. The correlations between the post-diet substrate oxidation parameters and the calpain activity in the 100% diet group are shown in Table 23. The post-diet calpain activity was most strongly correlated to the RQ value obtained 120 minutes post-prandially (beta = -3.56; p < 0.010), as compared to the correlation obtained 150 minutes postprandially (beta = 2.55; p < 0.020). Table 23: Post-diet substrate oxidation parameters correlated to the post-diet calpain activity in the 100% diet group (n = 7). Parameter  R Square  Adjusted R  B  Beta (B)  Probability  Square RQ, 120  0.721  0.434  -1874.665  -3.56  0.010  0.927  0.79.0  1639.186  2.66  0.020  minutes RQ, 150  minutes Constant = 173.880  107  RESULTS 9.  FACTORS RELATING TOCHANGES IN MUSCLE FUNCTION  A. MALES Simple linear regression analysis was performed on the calpain data and the muscle function parameters for the purpose of determining the extent to which muscle function (dependent variable) was affected by the myofibrillar calpain activity (independent variable). None of the pre-diet muscle function parameters were significantly linearly related to pre-diet calpain activity. The significant post-diet correlations between muscle function parameters and • calpain activity in the male subjects is displayed in Table 24. The eccentric average torque obtained at a movement velocity of 120 degrees/second and the eccentric peak torque were significantly correlated to the post-diet calpain activity in a positive fashion.  Table 24: Post-diet muscle function parameters correlated to post-diet myofibrillar calpain activity in male subjects (n = 4).  Muscle  Multiple R  function  Adjusted R  B  ft  Probability  0.977  8.547  0.99  0.0078  0.951  6.131  0.98  0.0164  square  parameter eccentric peak, 0.992 120 deg/sec eccentric  0.984  average, 120 deg/sec  .  108  RESULTS  The correlations between the change in muscle function parameters and the change in calpain activity in the male subjects is displayed in Table 25. Both the change in the eccentric average torque at a movement velocity of 120 degrees/second, and the eccentric peak torque at a movement velocity of 120 degree/second were positively related to the change in calpain activity.  Table 25: Correlation coefficients for change in calpain activity to change in muscle function parameters for the male subjects (n = 4).  Muscle  Multiple R  function  Adjusted R  B  ft  Probability  0.879  9.302  0.96  0.0412  0.859  6.558  0.95  0.0482  square  parameter eccentric peak, 0.959 120 deg/sec eccentric  0.918  average, 120 deg/sec  B.  FEMALES  Simple linear regression analysis was performed on the calpain data and the muscle function parameters for the purpose of determining the extent to which muscle function (dependent variable) was affected by the myofibrillar calpain activity (independent variable). Only the significant correlations between the pre-diet muscle function parameters and calpain activity are shown in Table 26. All pre-diet concentric torques were significantly correlated with pre-diet calpain activity except for the concentric peak torque at a movement velocity of 180 deg/sec and the concentric peak torque at a velocity of 90 deg/sec within the whole group of female subjects.  109  RESULTS  Table 26: Pre-diet muscle function variables correlated to pre-diet myofibrillar calpain activity in the entire group of athletic females (n = 14).  Muscle function parameter  Multiple R  Adjusted R B square  B  Probability  concentric  -0.697  0.440  -1.477  -0.70  0.0081  -0.665  0.391  -1.155  -0.66  0.0131  -0.662  0.388  -1.640  -0.66  0.0137  -0.685  0.416  -1.485  -0.68  0.0140  -0.640  0.356  -1.942  -0.64  0.0185  -0.635  0.349  -1.500  -0.63  0.0198  -0.620  0.329  -1.100  -0.62  0.0236  average, 120 deg/sec concentric average, 180 deg/sec concentric average, 90 deg/sec concentric peak, 120 deg/sec concentric peak, 90 deg/sec concentric peak, 30 deg/sec concentric average, 30 deg/sec  110  RESULTS Table 27 displays the correlations between muscle function parameter and calpain activity in the 100% diet group. All pre-diet concentric torque values were significantly correlated with pre-diet calpain activity in the 100% diet group, except for the following muscle function parameters where there was an nearly significant linear relationship between each one and pre-diet calpain activity: concentric average torque, 30 deg/sec (p = 0.0561); concentric average torque, 180 deg/sec (p = 0.0822). The only correlation between muscle function and pre-diet calpain activity within the 75% diet group occurred at a movement velocity of 180 deg/sec where the concentric peak torque was almost significantly correlated to the pre-diet calpain activity (r = -0.864; Adjusted r = 0.661; B= 2  -3.235; Beta = -0.86; p = 0.0591).  111  RESULTS  Table 27: Pre-diet muscle function variables correlated to pre-diet myofibrillar calpain activity in athletic females in the 100% diet group (n = 7).  Muscle  Multiple R  function  Adjusted R  B  ft  square  parameter concentric  Probability .  -0.798  0.564  -1.602  -0.80  0.0316  -0.779  0.528  -1.599  -0.78  0.0391  -0.756  0.486  -1.937  -0.76  0.0493  -0.742  0.461  -1.651  -0.74  0.0561  -0.740  0.456  -1.308  -0.74  0.0575  -0.730  0.439  -2.223  -0.73  0.0627  -0.718  0.418  -1.286  -0.72  0.0690  -0.696  0.382  -1.185  -0.70  0.0822  peak, 120 deg/sec concentric average, 120 deg/sec concentric average, 90 deg/sec concentric peak, 30 deg/sec concentric average, 30 deg/sec concentric peak, 90 deg/sec concentric peak, 180 deg/sec concentric average, 180 deg/sec  112  RESULTS All of the significant correlations (or correlations that were almost significant) between post-diet muscle function and post-diet calpain activity occurred witMi the concentric conditions for both diet groups. The correlations between the post-diet muscle function variables and post-diet calpain activity for the 100% diet group are shown in Table 28. There were significant correlations between the following post-diet muscle function parameters and the post-diet calpain activity for the 100% diet group: concentric peak torque, 90 deg/sec; concentric peak torque, 120 deg/sec; concentric average torque, 120 deg/sec.  113  RESULTS  Table 28: Post-diet muscle function variables correlated to post-diet myofibrillar calpain activity for athletic females in the 100% diet group.  Muscle function parameter  Multiple R  Adjusted R B square  ft  Probability  concentric  -0.873  0.714  -0.529  -0.87  0.0104  -0.816  0.598  -0.528  -0.82  0.0254  -0.813  0.594  -0.474  -0.81  0.0261  -0.704  0.3939  -0.428  -0.70  0.0778  -0.699  0.387  -0.519  -0.70  0.0804  peak, 90 deg/sec concentric average, 120 deg/sec concentric peak,120 deg/sec concentric average, 30 deg/sec concentric average, 180 deg/sec  114  RESULTS  The post-diet correlations between muscle function variables and post-diet calpain activity are shown in Table 29. The only significant between a muscle function parameter and post-diet calpain activity occurred with the post-diet concentric peak condition at a movement velocity of 120 deg/sec. Also included in Table 29 were correlations between post-diet muscle function parameters and post-diet calpain activity that were almost significant  115  RESULTS  Table 29: Post-diet muscle function variables correlated to post-diet myofibrillar calpain activity for athletic females in the 75% diet group.  Muscle function parameter  Multiple R  Adjusted R square  B  ft  Probability  concentric  -0.882  0.703  0.798  0.88  0.0480  -0.851  0.631  0.869  0.85  0.0678  -0.825  0.573  0.304  0.82  0.0859  -0.7300  0.416  0.559  0.73  0.0996  -0.711  0.381  0.369  0.71  0.1136  peak, 120 deg/sec concentric peak, 90 deg/sec concentric peak, 180 deg/sec concentric average, 120 deg/sec concentric average, 180 deg/sec  116  RESULTS  Table 29: Post-diet muscle function variables correlated to post-diet myofibrillar calpain activity for athletic females in the 75% diet group.  Muscle  Multiple R  function  Adjusted R  B  B  Probability  square  parameter concentric  -0.882  0.703  0.798  0.88  0.0480  -0.851  0.631  0.869  0.85  0.0678  -0.825  0.573  0.304  0.82  0.0859  -0.7300  0.416  0.559  0.73  0.0996  -0.711  0.381  0.369  0.71  0.1136  peak, 120 deg/sec concentric peak, 90 deg/sec concentric peak, 180 deg/sec concentric average, 120 deg/sec concentric average, 180 deg/sec  116  RESULTS  Change in muscle function parameters that were significantly correlated to the change in calpain activity for the female athletes occurred only in the 75% diet group and can be seen in Table 30. The only significant correlation occurred between the change in concentric peak torque at a movement velocity of 180 deg/sec and the change in calpain activity. There were almost significant correlations between the change in muscle function and calpain activity within the 75% diet group for the eccentric peak condition at movment velocities of 90 and 180 deg/sec (Table 30).  Table 30: Correlation coefficients for change in calpain activity to change in muscle function variables for the female athletes in the 75% diet group (n = 7).  Muscle function parameter  Multiple R  Adjusted R square  B  fi  Probability  ecccentric  0.936  0.844  -0.956  -0.94  0.0061  0.936  0.834  -0.352  -0.94  0.0194  0.756  0.464  -0.768  -0.76  0.0824  peak, 180 deg/sec concentric peak, 180 deg/sec ecccentric peak, 90 deg/sec  117  RESULTS 10. SUMMARY OF RESULTS WITH REFERENCE HYPOTHESES Hypothesis  TO THE STUDY  1:  Changes in fat oxidation, induced by dietary restriction and aerobic exercise, result in no difference in quadriceps muscle group function.  The null hypothesis was confirmed for both male and female subjects. Although there were selective changes in muscle function at specific movement velocities in both subject groups, there were no significant trends in the change in overall muscle function.  Hypothesis 2: Changes in fat oxidation, induced by dietary restriction and aerobic exercise, result in no difference in proteolysis of skeletal muscle.  The null hypothesis was generally confirmed as there were no significant changes in myofibrillar calpain activity in both the male and female subject groups. It is important to note however, that the small sample size, and the large inter-subject-variability (especially in the female subject group) renders the detection of small differences unlikely. It is also interesting to note that calpain activity was negatively correlated to a number of the muscle function parameters in the female subject group, suggesting that an inverse relationship exists between the two variables. A conclusive relationship between muscle function and calpain activity can not be made due to the fact that calpain activity was positively correlated to a number of muscle function parameters in the female subject group.  118  DISCUSSION  Chapter V DISCUSSION  /.  INTRODUCTION A brief review of the subjects involved in the study will introduce the discussion.  This will be followed by the major findings pertaining to each of the hypotheses: (i) changes in fat oxidation, induced by dietary restriction and aerobic exercise, as related to protease activity (protein degradation); (ii) changes in fat oxidation, induced by dietary restriction and aerobic exercise, as related to muscle function. Subsequently, the overall relationships established through this study will be reviewed, and lastly conclusions will be presented.  2. SUBJECTS A) MALES: In accordance with the subject criteria, the non-smoking male subjects were sedentary, as none of them had been regularly active within the last two years. Their lack of activity and trained status was evident by their low  max value for their age (which  was below the average value of 45 ml/min/kg found for men between the ages of 20 to 29 (Canadian Standardized Test of Fitness Operations Manual, 1987)). It was not indicative of a trained person (indicated by a V O 2 max value of greater than 51 ml/min/kg) (Canadian Standardized Test of Fitness Operations Manual, 1987). The subjects' lack of activity was also indirectly reflected by their high body weight which was 20% higher than their ideal body weight. The large body weight corresponded to a BMI that was outside the healthy limit of 18-25 kg/m (Hunt and Groff, 1990). This value fell into the range of BMI values used to 2  119  DISCUSSION 2 descnbe type I obesity (BMI = 25-29 kg/m ; Garrow, 1981). Lack of activity has been shown to be a contributing factor for obesity (Robinson et al, 1990). The average percent body fat of the male subjects was within the desired subject criteria range of 10-20%. Furthermore, this percent body fat indicated that the male subjects were in a range where optimal health was attainable (10-25%; Fox et al, 1993). 1) D i e t a r y I n t a k e : According to the subjects' 4-day diet records, the proportion of carbohydrate, protein, and fat consumed was approximately within the recommended healthy nutrient intakes (50-55% carbohydrate, 12-15 % protein, and 25-30% fat; Faber and Spinnler Benade, 1991). Analysis of usual intake (four-day food records) showed that energy intake supplied 175% of the recommended energy intake suggested for a 22 year old sedentary male of height and weight 176 cm and 84 kg respectively (Food Processor II Nutrient Analysis Software, E S H A Research, Salem, OR). The positive energy balance produced by the combination of the higher energy intake and low energy expenditure may explain why the subjects were classified as being type I obese. It is well known that the combination of a high energy intake and lack of activity are contributing factors to obesity (Robinson et al, 1990). It is also possible that the male subjects overestimated their usual intake. An intake above 75% of their recommended suggests that they would have been actively gaining weight, or exercising. The subjects did comment on the fact that they had gained a small amount of weight in the past year. Therefore, the possibility that they overestimated their energy intake is highly likely. The high consumption of meat could, in part, explain why the following nutrients exceeded the RNI, because meat (chicken, beef, pork, steak) contains a high proportion of at least 1 of these nutrients: protein, thiamin, niacin, vitamin B12, iron, phosphorus,  120  DISCUSSION selenium. The large amount of potassium and sodium consumed may have been due to the large consumption of salted food (fast foods, potato chips).  B. FEMALES Fourteen female subjects (7 subjects per group) were recruited for the study. In order to determine if this number was large enough to detect a difference in calpain activity, the average standard deviation associated with calpain activity obtained from the female subjects was inserted into the following sample size equation (Brown and Hollander, 1977):  Sample Size (n) = (2z x v )/ (Difference ) 2  2  where n = population size per groups; z = 1.96 so that all tests were done at a significance level of p < 0.05; Difference = 0.10; v = variability associated with determining calpain activity. The "Difference" term represents a value required in order to find a significant difference between each group. For this study, a difference in the amount of calpain activity of 10% was selected as meaningful, based on the study of Belcastro et al (1988). The variability term of 25 units/g wet weight was determined from the female subjects. Sample Size (n) = [2 (1.96) x 25 ] / (IO ) 2  2  2  Therefore, n = 48, and the total sample size = 96.  Thus, a larger sample size should have been recruited as the total subject number was to low to find a significant difference in calpain activity. In accordance with the subject criteria, all of the female subjects were runners who had indicated that they ran at least 50 km/week. Eleven subjects were competitively  121  DISCUSSION  involved in long distance running, and all of the subjects participated in at least 2 other types of exercise other than running. All of the subjects possessed V O ^ max values that were greater than the subject criteria of 42 ml/kg/min signifying that the subjects were well-conditioned females (Canadian Standardized Test of Fitness Operations Manual, 1986). Their commitment to athletics was high as they had been involved in running, and had been exercising regularly and/or competitively for the past 9.4 ± 1 . 0 years. Their dedication to athletics was also seen by the fact that they exercised approximately 100 minutes/day throughout the duration of the study. Eight subjects were taking oral contraceptives. This was the only type of medication the subjects were ingesting. Only subjects with regular menstrual cycles were accepted into the study due to the inherent difficulties associated with measurements of female athletes with oligomenorrhea or amenorrhea. All of the subjects were within the desired subject criteria age range (18-35 years) except for one subject, who was 47 years old. This subject was accepted into the study because of her exceptional athletic ability and fulfillment of all other subject criteria. The average percent body fat for all of the subjects fell within the desired subject criteria range of 16-26%. This value was in accordance with those previously found for female endurance athletes (Fox et al, 1993). The percent body fat for the athletes in both diet groups was slightly higher than that found for a group of 70 female distance runners, where the relative body fat averaged 16.8 ± 5.5 (Wilmore, 1977). However, a woman who established the best time in the world for a 50 mile run in 1987 had a percent body fat of 35.8%. Thus, the percent body fat is not the sole determinant of the caliber of the athlete. Comparisons of the demographic data demonstrated that the 100% and 75% diet groups were well matched with regard to age, height, weight, percent body fat, fat-free mass (FFM), and fitness level. The subjects described themselves as weight stable within  122  DISCUSSION the last 3 months. The subjects were also very well matched in terms of fat distribution in accordance with skinfold values.  i) Dietary Intake: The energy intake found in this study was 84 ± 6% (2052 ± 153 kcal/day) of the energy requirement in the subject group as a whole, when activity level was considered. Mulligan and Butterfield (1990) examined the energy intake of female runners, aged 19-41, and found that they met 75% (1974 kcal/day) of their energy requirement each day.  Other  researchers have further confirmed the finding of a low energy intake in runners: Clement and Asmundson (1982) found the mean energy intake in 17 female runners, aged 21-22 years, averaging 44 miles/week to be 2026 kcal/day; Lampe et al (1986) found that 9 female distance runners, aged 27-34, running 45 miles/week had a mean energy intake of 1922 kcal/day which was less than mean estimated energy expenditure per day (based on activity level); Prior et al (1990) found that female marathoners and recreational runners had mean energy intakes that were 1720 kcal/day and 1708 kcal/day respectively. Thus, according to four-day diet records of the subjects, the finding in the present study that energy intake was low in female participants of a sport such as running, where weight and/or body fat is an issue, is consistent with findings from other studies (Beals and Manore, 1994). An energy intake lower than the maintenance energy requirement may be explained in part by under-reporting of food intake (Livingston et al, 1990). For example, Mertz et al (1992) found that this underreporting represented 18-20% of energy required to maintain weight in a group of males. Edwards et al (1993) also found that women runners underreported energy intake, possibly from 4-58% of total energy requirement. In the group of female subjects, under-reporting of energy intake was highly possible because they had reported weight stability over the past 6 months. Factors affecting the incidence of under-reporting are not well known, nor have strategies to control these factors been successful (Block, 1982; Gibson, 1987; Mertz et al,  123  DISCUSSION  1992; Stunkard, 1981). Mertz et al (1992) suggested that in a group of female runners, under-reporting may have been subconscious and motivated by the belief that low body weight is accomplished through eating less. Many studies have reported the presence of physiological adaptations in athletes when net energy intake was negative due to the weight stability of the subjects. For example, Mulligan and Butterfield (1990) reported weight stability in very active runners over a two month period where 75% of the energy requirement was met. Prior et al (1990) found that recreational runners and marathoners had almost identical intakes (1708 and 1720 kcal/day respectively), yet the marathoners were running nearly three times the weekly mileage of the recreational runners and still reported stable body weights. It is questionable as to whether or not the athletes did actually ingest less food because the method used to determine energy intake varied between studies (Edwards et al, 1993; Schulz et al, 1992; Wilmore, 1991). However, it has been suggested that suboptimal energy intakes may be the result of physiological adaptations, specifically an increased energy efficiency, which allows some female athletes to function normally and maintain energy balance on fewer calories than would be expected based on body size and activity level (Clark et al, 1988; Thompson and Blanton, 1987). The percent macronutrients ingested in the subjects' diets were very similar to those recommended for healthy individuals (50-55% carbohydrate, 12-15% protein, and 25-30% fat; Faber and Benade, 1991). This is similar to the percentages previously found for male marathon runners (of which the percentage of calories from carbohydrate, protein and fat approximated the general US population; Rokitzki et al, 1994): 54% carbohydrate, 31% fat, and 12% protein. All subjects met the RNI for saturated and monounsaturated fat intake (10% of caloric intake; Nutrition Recommendations, Health and Welfare Canada). All subjects ingested slightly less than the RNI for polyunsaturated fats, where polyunsaturated fats  124  DISCUSSION were 7.1 ± 0.01% and 8.5 ± 0.02% of total caloric intake in the 100% and 75% diet groups respectively. All of the subjects in this study ingested lower than the recommended amounts of copper and pantothenic acid. The decreased intake of these nutrients has not been previously documented in athletes, and may have been due to the food types that the subjects chose to ingest. Thus, these findings may have occurred as a result of limitations of the Canadian Nutrient file for assessing intake of these nutrient accurately (Barr et al, 1994). The subjects in this study ingested large amounts of vitamin C , potassium and sodium. Potassium and sodium were probably ingested in large amounts to replace the electrolytes lost through exercise (Fox, 1984). The high vitamin C intake has been previously documented in athletes: Rokitzki et al (1994) found vitamin C intake to be 2.23 mg/kg body weight for a group of male marathon runners (compared to 2.61 mg/kg for this group of subjects); Faber and Benade (1991) found that a group of elite field athletes consumed 2.12 mg/kg body weight per day (corresponding to an absolute value of 179 mg/day).  3.  ANTHROPOMETRICS The amount of weight loss in the male subject group (1.5 ± 0.3 kg) over the 9 day  study period corresponded to a healthy amount of weight loss in the respective time period (Robinson et al, 1990). The amount of weight lost was larger than expected weight loss of 1.0 kg. The expected weight loss was determined by dividing the energy deficit created in nine days (7623 kcal) by the energy deficit needed to lose 1 kg body weight (7700 kcal/kg; Robinson et al, 1990). A larger amount of weight lost than expected was probably due to the loss of water that often accompanies dieting (Bailor, 1991).  125  DISCUSSION The amount of weight lost in the female subjects who were in the 75% diet over the two week diet period was 1.7± 0.3 kg and corresponded to a healthy amount of weight lost in the respective time period (Robinson et al, 1990). Studies illustrating the components of weight lost in athletes with an imposed energy restriction have not been documented. However, studies looking at weight loss in obese persons suggest that weight lost over short term could have been due to loss of water, fat, protein, and/or glycogen (Brownell et al, 1987). It has been determined that as the length and severity of the energy restriction increases, the contribution of fat stores to total weight lost increases substantially (Brownell et al, 1987). This is in contrast with results found from this study, where the loss of fat mass of the male subjects and the female subjects who were in the 75% diet group was a large contributor to the decrease in body weight occurring as a result of the imposed moderate energy restriction of relatively short length. These results are consistent with Bailor (1991) who found that a short term moderate energy restriction did result in a significant loss of fat mass in Sprague-Dawley rats, while a severe energy restriction did not. The food allotment of the moderately restricted (MR) and severely restricted (SR) groups was reduced by 25 and 50% respectively for 12 weeks during which they were fed a low-fat vitamin supplemented rat chow mix. The rats were exercised on a motor-driven treadmill set on a 15% slope at 12 m/min for 30 min/day, 5 days/week. There are however, limitations to the study because the control rats were placed on a high fat diet and the rats were still growing throughout the experiment. Also, application of these results to this study may be limited due the fact that Bailor's study used rats and the duration was short term. It is difficult to compare the loss of fat mass in the male subjects and the female subjects who were in the 75% diet group to the loss of fat mass found in other studies because of the differences in subject type (most studies look at the effect of an energy restriction on obese subjects) and severity and length of the energy restriction. Nonetheless, the change in fat mass which occurred in the male subjects and the female  126  DISCUSSION subjects in the 75% diet group appeared to contribute significantly to the decrease in body weight It appeared that more fat mass was lost than body mass lost This may have been due to errors associated with determining total body fat from skinfold measurements. Determination of total body fat from skinfold measurements is based on the assumption that a direct relationship exists between total body fat and subcutaneous fat. Errors in determining total body fat from skinfold measurements are associated with the validity of this relationship. For example, individuals distribute fat differently, where some may deposit fat more internally versus externally (subcutaneous fat). Therefore, a direct relationship between total body fat and sucutaneous fat may not be consistent among individuals of different sex, age, and race. Another factor that may contribute to the error associated with skinfold measurements in determining total body fat includes differences in skin thickness and compressibility among individuals and among different skinfold sites. Compressibility of the subcutaneous fat layer may differ between sites and between individuals because the subcutaneous fat layer is not constant. The decrease in total body fat measured by skinfolds may have been confounded due to hydration changes. The female subjects in the 75% diet group and the male subjects may have been more dehydrated at the end of the study as it has been suggested that a diet restriction could result in less replete glycogen stores and therefore less conservation of body water (glycogen is stored in conjugation with body water; Bailor, 1991). This lower conservation of body water in the male subjects and the female subjects who were in the 75% diet group, while keeping the other constituents stable, tends to increase the overall density of the body. Increasing the body density makes the body appear to contain less fat, suggesting that the final percent body fat measurement for the male subjects and the female subjects in the 75% diet group was smaller than the actual value. As a result, the post-diet total fat mass may have been underestimated in the male subjects and the female subjects who were in the 75% diet group.  127  DISCUSSION Overall, limitations associated with using skinfolds to assess fat mass, as well as changes in hydration status of male subjects and female subjects in the 75% diet group challenges the validity of the loss of a large proportion of fat mass from total weight lost with an imposed short-term energy restriction.  128  DISCUSSION  4. EXERCISE A) MALES The average energy expenditure throughout the study duration for 30 minutes of jogging was 288 ± 1 8 kcal/day. During exercise, the male subjects maintained the recommended aerobic training heart rate at a range of 60-80% of maximum heart rate (Fox et al, 1993), indicated to stimulate improvements in endurance or performance (Fox et al, 1993). The heart rate reserve remained fairly constant (80%) between 5 and 30 minutes of exercise, indicating that the intensity of exercise was constant within this time period.  B) FEMALES Although the distance run per week was less than the desired subject criterion of 50 km/week,the subjects were involved in various other activities such as biking and swimming. Their participation in other activities was evident from the large amount of time spent exercising and each subject did at least two activities per day. The average energy expenditure per day (when considering days 1 to 9 of the study) was 832 ± 97 kcal/day, with the average energy expenditures being quite similar between the 75% and 100% diet groups. The variability in energy expenditure each day was high. This was probably due to the difference in type, intensity, duration, and frequency of the exercise performed among subjects. Variability in reported energy expenditure may have also arisen through the difficulty associated with filling out the daily exercise regimen by the subject. For example, although the subjects specified the intensity of exercise performed daily, it may have been difficult to define intensity of exercise as being in one of three categories (defined on the exercise recording form as low, medium, and high according to the amount of effort exerted). Although the intensity of exercise was defined for the subjects on the  129  DISCUSSION exercise recording form and recorded by the subjects, in calculating the energy expenditures from the diaries, it was difficult to interpret what subjects considered to be low, medium and high intensity. An attempt to circumvent these problems and define the intensity of exercise involved having the subjects record their maximum heart rate during an exercise bout and comparing this heart rate value to the theoretical maximum heart rate for that subject. However, subjects did not always remember to do this. Also, different people have different maximum heart rate values. Thus, a value for maximum heart rate may not be predicted in an accurate manner from the equation 220-age (as was shown with the male subjects). The amount of energy expended through exercise was high for the female subjects in both diet groups. However, the accuracy of these values is questionable due to limitations associated with the exercise recording form and its interpretation.  5. SUBSTRATE OXIDATION There was no significant change in resting energy expenditure (REE) after diet and exercise treatment, even when corrected for body weight and fat-free mass (FFM) in both the male and female subjects. This is likely due to the short time period of the study. Furthermore, the sample size may have been too small to elicit a statistical difference in both male (n = 3) and female subjects (there were 7 subjects in each diet group). In the male subjects there was no significant change in the resting RQ value as a result of the diet and exercise treatment. This signifies that there was no change in substrate oxidation at rest as a result of this treatment. However, the applicability of the results are questionable due to the small sample size. The female subjects in the 7 5 % diet group, on the other hand, had a significant decrease in the resting RQ value. This means that there was a change in substrate utilization in the female athletes as a result of the imposed energy restriction such that fat  130  DISCUSSION was utilized to a larger extent under resting conditions compared to the pre-diet value. This is consistent with results from Froidevaux et al (1993) who found that the resting R Q value decreased from 0.86 ± 0.03 to 0.77 ± 0.02 in 10 moderately obese women. The results obtained from the female subjects in the 75% diet group correspond with those obtained from Nelson et al (1992) who found that there was a significantly larger percent of resting energy expenditure derived from lipid after a 5 month diet restriction in obese females. However, the time period of these two studies is very different. The results obtained from the female subjects, concurrent with results obtained from other studies, demonstrate that an energy restriction created by diet and exercise promotes the utilization of fats as an energy source under resting conditions. Both the male and female subjects experienced a change in substrate oxidation after the consumption of a standard test breakfast. Specifically, for the male subjects there was a significant decrease in the R Q value at 60,120, and 150 minutes post-prandially. The applicability of these results may be questionable, however, again due to the small sample size. The female subjects in the 75% diet group had significantly lower R Q values at 30, 120 and 150 minutes post-prandially as compared to their pre-diet value and compared to the change in the 100% diet group. Although the subject size of the male group limits the applicability of the results, the similar decreases in RQ values observed for males as was seen for the females indicated that fat utilization was elevated post-prandially as a result of the diet and exercise program. The decrease in the RQ value at these time points may have also been due to the lower energy value of the meal that was fed post-diet to the 75% diet group. However, the significant decrease in the resting RQ value suggests that the decrease in RQ values is an effect of the diet, and not the meal. These results are consistent with findings of Froidevaux et al (1993) who found that the post-absorptive RQ decreased from 0.81 ± 0.04 to 0.76 ± 0.03 in 10 moderately  131  DISCUSSION obese women who lost weight as a result of a diet and exercise program. They also measured the rate of lipid, carbohydrate and protein oxidation before and during the weight loss period for 24 hours in a respiratory chamber and found that the rate of lipid oxidation was significantly increased as a result of weight loss (from 84 ± 31g/day to 141 ± 33 g/day).  6. MUSCLE  PROTEIN  DEGRADATION  To date, calpain activity has never been documented in human subjects. It is interesting to note in the present study that the myofibrillar calpain activity of the trained female athletes was about three times the value found for the male subjects, both pre- and post-diet. This elevated activity in the female athletes may have been due to a training effect on calpain activity. It has previously been demonstrated that with high volume exercise, calpain activity increases with increasing intensity (Belcastro, 1995). Calpain activity in the plantaris muscle (which contains predominantly fast fiber types) of rats doubled when intensity was increased from 55% VC«2 max to 75% V O ^ max (Belcastro, 1995). Thus, in human muscle containing mixed fiber types, such as the vastus lateralis (the muscle in which calpain activity was assessed), the training program (involving high volume and high intensity exercise) in the female subjects may have caused the calpain activity to be elevated above that found for the male subjects. Reasons for the increased calpain activity in the trained female subjects may be due to: the regulation of phosphorylation of myosin light chain (through the interaction of myosin light chain kinase and calpain); regulation of glycolytic enzymes; myoblast rearrangement to accommodate fusion (through the action of calpain on glycoproteins; Bond and Butler, 1987); regulation of phospholipid turnover (due to the membrane being a site for calpain activation (Johnson, 1990)); modulation of critical transport processes of the membrane (Johnson, 1990); post-translational modification of proteins/enzymes (Johnson, 1990).  132  DISCUSSION The effect of an energy restriction on myofibrillar calpain activity has also not previously been investigated in human subjects. In this study, an imposed energy restriction in the form of diet and exercise appeared to increase in myofibrillar calpain activity (half fold from the pre-treatment activity in the male subjects, two fold from the pre-diet value in the female subjects who were in the 75% diet group). However, these changes in activity remained statistically insignificant in both the female and male subjects. In both subject groups, this may have been due to the small sample size. The absence of a significant change in myofibrillar calpain activity in the female group of subjects may have been due to high variability in calpain activity. The large standard deviations associated with the calpain activity of the female subjects may be related to the exercise regimen performed by each subject. Female subjects were instructed to exercise "as usual". Differences in intensity, time, frequency, and type of exercise performed may have been responsible for the large variability in myofibrillar calpain activities. The variability associated with exercise intensity has recently been documented, where calpain activity increased with increasing intensity in the plantaris muscle of rats (Belcastro, 1995). This intensity dependent increase in calpain activity was volume dependent, where the intensity effect was not seen when exercise volume was decreased. The type and frequency of exercise performed may also affect calpain activity, although the effect of these factors has not been previously documented. Thus, by controlling the exercise intensity, time, frequency, and type of exercise a lower variability in myofibrillar calpain activity may have been produced, and a statistical difference may have been possible. In the male subjects, exercise intensity, duration, frequency, and type was controlled. This was associated with a much lower variability in myofibrillar calpain activity, such that the post-treatment variability was almost five times lower in the male subject group compared to the post-diet variability in the female subject group.  133  DISCUSSION Although the subjects were instructed not to exercise 24 hours before their muscle biopsy, the timing of the muscle biopsy relative to the last bout of exercise may have also contributed to the variability in calpain activity in both the female and male subjects. For example, a higher calpain activity may have been detected if a muscle biopsy was taken 24 hours after exercise compared to the activity obtained with a biopsy that was taken 48 hours after exercise. It is unknown how long calpain activity remains elevated post-exercise. Thus, more controlled and standardized biopsy times may have decreased the variability in calpain activity. Moreover, the site chosen for the biopsy within the vastus lateralis may have introduced variability into the calpain activity in both groups of subjects. Although the variability in calpain activity at different sites in the vastus lateralis has not been documented, it has previously been suggested that fast muscle fibers may be more predisposed to changes in calpain activity compared to slow muscle fibers (Belcastro, 1995). For example, Belcastro (1995) has previously shown that the exercise intensity and duration effects on calpain activity are only seen in the plantaris muscle in the rat, which consists predominantly of fast fibers, while no effect was seen in the rat soleus muscle (consisting of predominantly slow fibers). Therefore, because fiber types are distributed unevenly in different parts of the vastus lateralis (superficial versus central, for example), the imposed energy restriction may have induced changes at sites that were different from the site chosen to assess activity. Thus, localized changes in myofibrillar calpain activity may or may not reflect changes in calpain activity within the whole muscle. The absence of a significant increase in myofibrillar calpain activity with an imposed energy restriction in both female and male subjects is in contrast to previous results in animals where an increase in myofibrillar calpain activity was observed in calorically restricted and fasted rats (Murray et al, 1991). Calpain activity was measured in hind leg skeletal muscle of mature Wistar rats, divided into four groups: control rats; 50% diet restriction (DR) over 48 hours; 75% DR over 48 hours; fasting for 48 hours. Calpain  134  DISCUSSION  activity, expressed as units per gram of muscle, was significantly increased in the 75% dietary restricted group and the fasted group compared to the controls. The calpain activity increased as the severity of the restriction increased (Murray et al, 1991). Although this study showed that myofibrillar calpain activity is very sensitive to the dietary regimen, it also showed that a more severe energy restriction may be required to elicit changes in the calpain activity. Murray et al (1991) found that the increase in myofibrillar calpain activity of the 50% DR group was not significantly different from the controls. Thus, the 33% and 25% energy restrictions imposed on the male and female subjects respectively, may not have been severe and/or long enough to create the significant changes in myofibrillar calpain activity as seen in the study by Murray and co-workers (1991). Results from this study are consistent with Kettelhut et al (1994) who found that activation of the calcium dependent pathway involving calpain was not responsible for the increased muscle proteolysis observed during fasting. They suggested that the increase in muscle proteolysis during fasting was attributable to an enhancement of the energyrequiring process by finding an increase in the ATP-dependent proteolytic pathway 1 day after food restriction in the extensor digitorum muscles of rats. Thus, the energy restriction imposed on the male and female subjects in this study may have activated other pathways involved in muscle proteolysis other than calpain. Attaix et al (1994) and Mitch et al (1994) suggest that the ATP-dependent pathway involving ubiquitin and proteasomes is responsible for the enhanced degradation of muscle proteins as a result of energy restriction. This was concluded by observing that under conditions where intracellular ATP was depleted, the increased proteolysis due to fasting was almost completely suppressed (Medina et al, 1992; Wing and Goldberg, 1993). The rise in ATP-dependent proteolysis observed during starvation was associated with increased mRNA level for ubiquitin and the 14 kDa ubiquitin-conjugating enzyme (which mediates the formation of ubiquitin-protein conjugates), accumulation of ubiquitinated  135  DISCUSSION proteins, and increased expression of subunits of the 20S proteasome (Medina et al, 1992; Wing and Goldberg, 1993). Upon refeeding, total and ATP-dependent proteolysis, levels of ubiquitin-protein conjugates, and expression of ubiquitin (Medina et al 1992), the 14 kDa enzyme (Wing and Banville, 1994), or proteasome subunits (Medina et al, 1992) returned to normal. Adenosine triphosphate-ubiquitin-stimulated proteolysis was also enhanced in the soluble extracts of skeletal muscle from fasted rabbits (Medina et al, 1992), in accordance with an increased mRNA level for the proteasome subunit. Results from the current study are in contrast to those found by Fagan et al (1992) who suggested that the increase in myofibrillar protein degradation observed during an energy depleted state was associated with an increase in calpain activity. This was determined by observing that trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (also known as E64; a compound that inhibits the calpains and the lysosomal cysteine proteases) in combination with calcium resulted in a decrease in myofibrillar degradation in energy depleted rats. Because there was no significant increase in calpain activity post-treatment or postdiet in either group of subjects it is also possible that myofibrillar protein degradation did not change as a result of the imposed energy restriction. The lack of a significant increase in myofibrillar protein degradation with an imposed energy restriction is consistent with prior results where an increase in myofibrillar protein degradation was not observed in rats who engaged in an exercise and a food restriction program or an exercise program (Kasperek et al, 1992). Rats were starved for 24 hours before exercise on a treadmill for 200-215 minutes and then they were re-fed ad libitum the day after exercise. All rats had pair-fed controls. Myofibrillar protein degradation was determined by the measurement of the rates of release of 3-MH from the perfused single Sprague-Dawley rat leg and was measured immediately after and 1 day following the exercise bout The findings of Kasperek et al (1992) were mdirect, such that re-feeding after 1 day of starvation, resulted in an increased food consumption which obliterated the increased  136  DISCUSSION protein degradation seen in rats who were fed before the exercise bout. Kasperek et al (1992) suggested that exercise prevents an increase in the rate of protein degradation caused by fasting. The mechanism responsible for this exercise-induced muscle protein sparing is unknown, but a conservation mechanism was implicated where muscle proteins are conserved when energy reserves are low (Kasperek et al, 1992). It is difficult to make conclusions about the effect of an energy restriction on muscle protein degradation because the assessment methods of different studies, measure different aspects of myofibrillar protein degradation. For example, 3-MH reflects actin and myosin degradation, while calpain degradation reflects the degradation of the cytoskeletal and sarcolemmal proteins. Thus, further research is needed to find a method that accurately reflects myofibrillar protein degradation and to elucidate whether calpain degradation rates and 3-MH excretion represent myofibrillar protein degradation rates. A method of assessing muscle protein degradation would allow the effects of an energy restriction on muscle protein degradation to be clarified.  7.  MUSCLE FUNCTION Peak torque is often the parameter used to describe muscle strength. The peak  torque values for the active subjects are unique in that they include both eccentric and concentric strength scores, whereas usually only concentric strength scores are presented (Snow and Blacklin, 1992). The value most often reported in the literature is the concentric knee extension torque at a slow movement velocity (usually 30 degrees/second) and a fast movement velocity (usually 180 degrees/second or 230-250 degrees/second). The pre- (163 ± 24 Nm; mean, SEM) and post-diet (157 ± 10 Nm; mean, SEM) concentric peak torque values for the male subjects at a movement velocity of 180 degrees/second were larger than the value of 117 ± 23 Nm (Kannus and Beynnon, 1993) found for forty-three healthy males (178 ± 7 cm; 76 ± 8 kg; 29 ± 5 years). The pre- and  137  DISCUSSION post-diet concentric peak values at a movement velocity of 30 degrees/second were lower than that found for elite male sprinters (267 ± 42 Nm; Alexander, 1990), as was expected. The concentric peak torque values at a movement velocity of 30 degrees/second corrected for body weight pre- (1.89 ± 0.22 Nm/kg body weight) and post-diet (1.92 ± 0.11 Nm/kg body weight) at a movement velocity of 180 degrees/second were also lower compared to corrected torque value obtained for elite male sprinters (3.65 ± 0.6 Nm/kg body weight; Alexander, 1990). These values are lower than that found for physical education majors of 3.0 ± 0.7 Nm/kg body weight at 30 degrees/second (Smith, 1987). The pre- and post-diet corrected and absolute eccentric peak torque values at a movement velocity of 30 degrees/second for the male subjects were lower than those found for elite male sprinters, (279 ± 57 Nm and 3.83 ± 0.8 Nm/kg body weight; Alexander, 1990). It was expected that the male subjects would have lower strength values than their athletes, as they were quite inactive. The pre- and post-diet concentric peak torque values for the female subjects in the 100% (118 ± 9 Nm and 124 ± 8 Nm respectively, mean ± SEM) and 75% (116 ± 8 Nm and 109 + 0 Nm respectively, mean ± SEM) diet groups at a movement velocity of 30 degrees/second were lower than those found for elite female sprinters (171± 40 Nm; mean, S E M ; Alexander, 1990) and slightly higher than those found for nonathletic females (96 Nm; Snow and Blacklin, 1992). The pre-diet corrected concentric peak torque at a movement velocity of 30 degrees/second (where torque was divided by body weight) values in the female subjects who were in the 100% (2.01 ± 0.15 Nm/kg body weight) and 75% (1.79 ± 0.12 Nm/kg body weight) diet groups was much lower than that found for elite female sprinters (2.95 Nm/kg body weight) for a knee extension (Alexander, 1990). This was expected as the subjects in this study were endurance athletes, not power athletes. It has previously been reported that endurance athletes had significantly lower concentric peak torque values at movement velocities ranging from 50 degrees/second to 300 degrees/second (Taylor et al,  138  DISCUSSION 1990). Thus, overall the female subjects had pre- and post-diet concentric peak torque values that were better than the average female, but were not as high as those reported for elite athletes. The corrected pre- and post-diet eccentric peak torque values obtained at a movement velocity of 30 degrees/second for the female subjects who were in the 100% diet (pre-diet, 1.90 + 0.03 Nm/kg body weight; post-diet, 2.07± 0.04 Nm/kg body weight) and the 75% diet groups (pre-diet, 1.72 ± 0.04 Nm/kg body weight; post-diet 2.00 ± 0.03 Nm/kg body weight) were also higher than the value of 1.52 ± 0.03 Nm/kg body weight previously found for 12 healthy females (Snow and Blacklin, 1992). Eccentric peak torque values could not be compared to those found for elite athletes as this parameter has not been frequently reported. The only significant decreases in muscle function post-treatment in the male subject group occurred with the concentric average contractions at a movement velocity of 30 degrees/second. The only significant decrease post-diet in peak torque for the female subjects occurred with the concentric average contractions in 75% diet group at a movement velocity of 120 degrees/second. The corrected post-diet concentric average torque at a movement velocity of 30 degrees/second was also significantly lower for the 75% diet group compared to the 100% diet group. These decreases in quadriceps muscle torque, post-diet in the female subjects who were in the 75% diet group, were accompanied by an increase in function at a movement velocity of 180 degrees/second where the post-diet eccentric average torque was significantly larger than the pre-diet value. This change in strength was unexpected. This increase in post-diet eccentric average torque at a movement velocity of 180 degrees/second for the female subjects in the 75% diet group may have been due to a greater learning effect of this group after the initial testing period. Additionally, the female subjects in the 100% diet group also showed increases in muscle torque at the completion of the diet treatment, where the post-diet eccentric and concentric average torques at  139  DISCUSSION movement velocities of 90 and 30 degrees/second respectively were significantly larger than the pre-diet value. This may have also been due to a learning effect. In the 75% diet group, this learning effect may have been due to the fact that at a faster movement velocity, one cycle takes much less time (0.73 seconds) than at 30 degrees/second (4.35 seconds), making it more difficult for the subject to learn to integrate sensory feedback with motor output (Snow and Blacklin, 1992). Therefore, subjects may have had greater difficulty producing a smooth, uninterrupted movement at maximal effort at the faster velocity, especially during the eccentric phase of muscle contraction. This phenomenon has previously been reported (Snow and Blacklin, 1992) where a movement velocity of 180 degrees/second gave the lowest within session (r= 0.92) and between session (r = 0.75) correlations compared to those found for a movement velocity of 30 degrees/second. In fact, the eccentric torque at a movement velocity of 180 degrees/second had the highest intra-subject coefficient of variation (8.9%) compared to other eccentric and concentric torque values obtained at movement velocities of 30 and 180 degrees/second (Snow and Blacklin, 1992). Therefore, additional practice may have been required for the female subjects in the 75% diet group to learn to perform as well as they did at the slower velocities. It is also possible that the increases in muscle torque post-diet in both the female and male subject groups represented training effects. It has previously been shown that the combination of resistance strength training and a very low calorie diet results in increases in strength (Pronk et al, 1992). Overall, the physical performance measures were not drastically impaired in either the male subjects or the female subjects within or between any dietary intervention group after weight loss. There were no significant trends in the absolute or corrected torque values pre- and post-diet for the male subjects and within each diet group for the female subjects. Furthermore, there were no trends in differences between the diet groups for the female subjects at the two time points. Thus, muscle strength and muscle performance was  1 4 0  DISCUSSION relatively unimpaired after 10 days of an energy restriction in both male and female subjects The fact that there was no overall decreases in muscle function in the female and male subject groups with an imposed energy restriction has been previously documented. Scott et al (1992) looked at the effects of a moderate caloric restriction on maximal physical performance in 36, mildly obese (30-40% body fat), premenopausal women (age 29-49 years). They consumed 1000 kcal/day with a diet composition of 20% fat, 60% carbohydrate, and 20% protein for 8 weeks. Muscle strength was assessed using a Cybex 340 isokinetic device during knee flexion and extension at a movement velocity of 60 degrees/second (Scott et al, 1992). Strength was determined by taking the highest torque value from one of three concurrent maximal efforts. Values for the pre- and post-diet measurements for eccentric (pre-diet, 137 ± 18 Nm; post-diet, 125 + 20 Nm) and concentric (pre-diet, 80 ± 12 Nm; post-diet, 79 ± 14 Nm) peak torques were not significantly different. Scott et al (1992) suggested that in order to induce any negative changes in maximal physical performance measures, either one or more of the following must occur: a greater degree of caloric restriction is required; or a longer dietary period is needed. Thus, the absence of a change in muscle function in the male and female subject groups with an imposed energy restriction in this study may have been due to the fact that: the energy restriction was not severe enough to induce changes in muscle function; or the 10 day imposed energy restriction period was not long enough to induce changes in muscle function. However, Pronk et al (1992) imposed a severe caloric restriction (intake was 520 kcal/day) with and without an endurance exercise program for a long period of time (90 days) and found no change in strength within and between groups. Strength was evaluated using the one-repetition maximum method with knee extension and knee flexion. The subject population consisted of severely obese females (n = 109). Thus, the applicability of results found by Pronk et al (1992) to the results of this study may be questioned.  141  DISCUSSION Nonetheless, this study did demonstrate that a severe energy restriction imposed over a long period of time did not result in changes in muscle function. Similarly, Fogelholm et al (1993) found that muscle function did not change in athletes following a 3 week gradual weight reduction program. Physical performance was assessed in 7 male wrestlers and 3 judokas using a 30-m sprint, vertical jumping height, and anaerobic performance. The 3 week diet restriction period involved a 1000 kcal/day decrease in energy intake by the subjects. There was a 2.7 ± 0.5% decrease in weight at the end of the diet period, which was similar to the weight loss experienced by the 75% diet group (2,6 ± 0.4%) in this study. The unchanged or even improved jumping height results indicated the preserved capability of the neuromuscular system to produce force. The results even suggested slightly better muscle mechanical function after the gradual weight loss program. Results from this study are not totally comparable to the results obtained from the female athletes because: different performance assessments were used; different athlete types were used (endurance versus more of a strength athlete); different diet protocols were used to induce weight loss, where the percent macronutrients and energy intake were not directly controlled. Results from the current study are in contrast with those found by Horswill et al (1990) who found that physical performance was impaired as a result of rapid weight loss. Physical performance was evaluated in 12 highly trained collegiate wrestlers both before and after the 4-day period of a hypocaloric low carbohydrate diet (11.4% protein, 46.7% fat, and 41.9% carbohydrate) with eight 15 second intervals of maximum effort sprints. After a 6% weight loss, there was nearly a three fold decrease in sprint work. It was suggested that the impaired performance after rapid weight loss was due to an alteration in blood volume (due to water loss) or an inhibition of glycolysis due to elevated free fatty acid levels in the blood. Results from this study are not completely comparable to the results obtained from the current study due to the fact that weight loss was rapid, and due to the fact that the ingested diet was high in fat.  142  DISCUSSION It is also possible that the imposed energy restriction in the female and male subjects resulted in a decrease in aerobic capacity, without a subsequent change in muscle strength. For example, Bender and Martin (1986) found that there was a decrease in endurance in 12 healthy females consuming an energy intake at 40% of normal daily intake. Endurance was assessed by measuring time to volitional exhaustion while walking on a treadmill that increased grade by 3% every 3 minutes. The time to volitional exhaustion was shorter following caloric restriction than after the normal diet (p < 0.001). Thus, the effect of an energy restriction may affect performance by decreasing endurance. This question was not evaluated in this study and deserves further attention. Overall, the fact that there was no change in muscle function with an imposed energy restriction is consistent with results obtained from many studies. However, comparisons made with other studies are limited by: the subject population; the diet and/or exercise protocol utilized; the severity and duration of energy restriction. Results obtained from this study are also limited by factors associated with using the Kin/Com isokinetic dynamometer to assess muscle function. For example variability may have been introduced into the measured torque values as a result of body stabilization, axial alignment, subject motivation, and the subject skill level. Although care was taken to align the knee axis with the machine's exercise-arm axis, the exact positioning was not precise, and there was a tendency for the subject's thigh to come forward during a test. Axial alignment, therefore, likely accounts for a significant portion of the variance.  143  DISCUSSION  8. POSSIBLE  RELATIONSHIPS  ELUCIDATED  FROM  THIS  RESEARCH  Two main associations were evaluated in order to test the hypothesis that substrate oxidation changes induce changes in calpain activity, which subsequently causes muscle function to change: 1) the effect of substrate oxidation on calpain activity 2) the effect of calpain activity on muscle function  A) SUBSTRATE OXIDATION AND CALPAIN ACTIVITY When stepwise regression analyses were performed on calpain activity and substrate oxidation measures (female subject group only). Significant relationships between calpain activity and substrate oxidation measures in the female subject group were not established pre-diet However, significant relationships were established between the post-diet calpain activity and the R Q values obtained 120 minutes and 150 minutes post-prandially. Calpain was inversely related to the RQ obtained 120 minutes post-prandially and was proportionally related to the RQ obtained 150 minutes post-prandially. This means that as the post-diet calpain activity increased the RQ obtained 120 minutes post-prandially decreased, while the R Q obtained 150 minutes post-prandially increased. A decrease in R Q at 120 minutes would indicate an increase in fat oxidation, while an increase in R Q at 150 minutes would indicate a decrease in fat oxidation. The post-diet calpain activity was determined to a larger extent by the R Q obtained 120 minutes post-prandially, compared to the RQ obtained 150 minutes post-prandially, as was evident by the larger standard correlation coefficient obtained at 120 minutes (-3.56; p = 0.010) compared to 150 minutes (2.66; p = 0.020). However, the coefficients are close in value, which signifies that although R Q 120 is more substantial in determining post-diet calpain activity, R Q 150 is almost equally contributive to determining the value of the post-  144  DISCUSSION diet calpain activity. Thus, there was no consistent increase in fat oxidation which causally influenced increased calpain activity. Furthermore, the change in RQ obtained 60 minutes post-prandially which was positively correlated with the change in calpain activity within the 75% diet group suggests that as fat oxidation decreased (signified by an increase in the R Q value) the calpain activity increased. Therefore, it is not known whether an increase in fat oxidation results in an increase in calpain activity due to the conflicting results obtained. However, it seems likely that changes in substrate oxidation do not change calpain activity as the significant increase in fat oxidation associated with an imposed energy restriction in this study was not consistently associated with an increase in calpain activity. Overall, the relationship of substrate oxidation and calpain activity remains to be elucidated. Further research is needed to investigate changes in substrate oxidation induced by varying the severity/intensity of diet and/or exercise protocols respectively and the subsequent changes in calpain activity.  B) MUSCLE FUNCTION AND CALPAIN ACTIVITY There was no overall change in muscle function or in calpain activity. In fact, the exact relationship between these two variables is inconclusive due to the differing results obtained with the male and female subjects. For example, significant positive correlations (obtained through simple linear regression between muscle function parameters and calpain) of the post-treatment calpain activity with eccentric average and peak torques at a velocity of 120 degrees/second for the male subject group suggests that muscle function increases with an increase in calpain activity. The changes between these variables for the male subject group were also significant and were directly proportional to one another.  145  DISCUSSION However, significant correlations (obtained through simple linear regression between muscle function parameters and calpain) between calpain activity and muscle function for the female subjects were negative signifying that as calpain activity increased, muscle function decreased, which was the hypothesized relationship. Most of the significant correlations between calpain activity and muscle function parameters occurred pre-diet and with concentric contractions. In fact, all of the pre-diet concentric average and peak torques were significantly correlated with the pre-diet calpain activity in the whole group of subjects, while only the pre-diet eccentric peak torques were significantly correlated to the pre-diet calpain activity. Although the correlations between the pre-diet concentric contractions and calpain were all highly significant, the pre-diet calpain activity was most strongly related to: concentric average torque, 120 degrees/second (beta = -0.70; p = 0.0081); concentric peak torque, 120 degrees/second (beta = -0.68; p = 0.0140); concentric average torque, 90 degrees/second (beta = -0.66; p = 0.0137); concentric average torque, 90 degrees/second (beta = -0.66; p = 0.0131). The only significant correlations that occurred between post-diet muscle function parameters and post-diet calpain activity for the female subjects occurred with the concentric contractions in the 100% diet group. Similar to pre-diet correlations, a high calpain activity was associated with a low torque value. The post-diet calpain activity in the 100% diet group was most strongly related to: concentric peak torque, 90 degrees/sec (beta = -0.87; p = 0.0140); concentric average torque, 120 degrees/sec (beta = -0.82; p = 0.0254); concentric peak torque, 120 degrees/sec (beta = -0.81; p = 0.0261). A highly significant negative correlation between the change in concentric peak torque at a movement velocity of 180 degree/second and calpain activity within the 75% diet group revealed that the change in calpain activity and muscle function assessed at a high movement velocity were inversely related. Because more tension per motor unit is generated with eccentric contractions (Vander et al, 1990), it was thought that the effect of changes in sarcomere structure  146  DISCUSSION induced by calpain may have been more profound in eccentric versus concentric contractions. However, this effect was not seen in the current study as the correlation between calpain activity and eccentric contractions were much less frequent. This may have been due to the fact that results obtained from eccentric contractions were more erratic, as the subjects found that the downward phase of contraction was more difficult to perform maximally. The greater amount of difficulty associated with producing smooth, uninterrupted movement during the lengthening phase of the quadriceps muscle group contractions has previously been documented in a group of 12 healthy women (Snow et al, 1992). The extent to which whole quadriceps muscle group function is determined by calpain activity evaluated in a small sample of muscle (15-35 mg) is questionable. For example, calpain may have induced localized changes in muscle function within the vastus lateralis for which the vastus lateralis muscle may have been able to compensate. It is unknown: if calpain activity varied within the muscle; if calpain produced localized or whole muscle functional changes; or if the whole muscle was able to compensate for localized changes in calpain activity if they did occur. Although an inverse relationship is suggestive between myofibrillar calpain activity and quadriceps group muscle function in the female subject group, a directly proportional relationship is suggestive in the male subject group. Therefore, the relationship between calpain activity and muscle function is inconclusive. The lack of a significant decrease in quadriceps muscle group function and change in myofibrillar calpain activity with an imposed energy restriction in the female subject group could have been due to metabolic adaptations in muscle protein structure previously present before the period of diet manipulation. The fact that these women had been competitively involved in endurance sports for the past 9.4 ± 1 . 0 years may have induced the myofibrillar protein adaptations allowing them to maintain muscle function with a succeeding imposed energy restriction.  147  DISCUSSION The reasons for the discrepancies between the results obtained from the males and the females may be due to the difference in sample size. Furthermore, the difference in the exercise regimen performed by the male and the female subjects may have affected the relationship as the exercise regimen performed by the female subjects was much more intense and of longer duration than that performed by the male subjects. The total energy deficit created in the male subject group may not have been large enough to produce changes in muscle function and/or calpain activity. Additionally, the difference in training status between the males and the females may have influenced the relationship between calpain and muscle function. In summary, more research is required to define the relationship between myofibrillar calpain activity and muscle function under well-controlled conditions.  9.  FURTHER  RESEARCH:  Future research is required on:  1. substrate oxidation and calpain activity, to establish a more specific relationship between the two factors. This may be accomplished by increasing the severity and length of an imposed energy restriction. 2. exercise and calpain activity, where the effects of endurance exercise frequency, type, intensity and duration need to be established. 3. exercise and calpain activity, where the length of time in which calpain activity is modified post-exercise needs to be established. 4. dietary/exercise factors related to calpain activity such as lean body mass, training status, athlete type (strength or endurance), etc. 5. establishing the reliability and validity of the calpain assay so that the variability in calpain activity can be established.  148  DISCUSSION  6. the variability in calpain activity at different sites of a whole muscle and evaluation of calpain activity obtained from a muscle sample and whole muscle calpain activity. 7. the difference in calpain activity between males and females. 8. the relationship between calpain activity and 3-MH release to determine if selective protein degradation rates are proportionally related to contractile protein degradation rates (assessed through 3-MH) for muscle and whole body changes. 9. developing a procedure for the assessment of skeletal muscle protein degradation rates of selective proteins.  10.  STUDY  LIMITATIONS  1. The lack of control of energy expenditure by the female subjects may have been responsible for producing the large variability in calpain activity. Control of the frequency, time, type, and intensity of exercise of the female subjects may have introduced less variability in calpain activity, and therefore enhanced statistical power. 2. The lack of control of the timing of the muscle biopsy may have also contributed to the variability in calpain activities, as some subjects may have exercised 24 hours before the biopsy, while others may have been resting for more than 24 hours. 3. Lack of conclusive knowledge as to whether myofibrillar calpain activity obtained from a small muscle sample reflects the overall calpain activity of the whole muscle. 4. Lack of validity of the calpain assay used to assess calpain activity in the vastus lateralis in human subjects. 5. Relating structural changes at the level of the myofibril to whole muscle group function. 6. Small sample size.  149  CONCLUSION  CONCLUSION  This study initially set out to determine if an imposed energy restriction in the form of diet and exercise resulted in an increase in fat oxidation, which subsequently may have increased calpain activity, which subsequently may have decreased muscle function. Overall, two relationships were established throughout this investigation: 1) an imposed energy restriction resulted in an increase in resting fat oxidation in the female subjects (nonsignificance in the male subjects). 2) high calpain activity was correlated with low muscle function for some parameters (only in female subjects).  The results from this research support the concept that an imposed energy restriction results in an increase in fat oxidation. This was evident by the decreases in the post-prandial R Q values as a result of the imposed energy restriction in both male and female subjects and a decrease in the resting RQ value for the female subjects. Additionally, it was inconclusive as to whether the changes in substrate oxidation induced by the energy restriction resulted in increases in calpain activity. The relationship of calpain and substrate oxidation as well as other factors that may be related to calpain activity (muscle damage for example) still requires further investigation. Although there were selective changes in muscle function, there was no change in quadriceps muscle function due to the lack of consistent trends within both sets of data. Increases in muscle torque observed in the female subjects were most likely due to a learning effect. The fact that myofibrillar calpain activity did not change with an imposed energy restriction indicated that a calpain induced activation of muscle proteolysis did not occur, aUuding to the idea that another mechanism may be involved in increasing muscle proteolysis (if increased at all).  150  CONCLUSION However, involvement of calpain cannot be completely ruled out. Perhaps the energy restriction was not severe enough or of long enough duration. Furthermore, characteristics of the female subjects (athletically trained; previously restricting their energy intake) may have induced myofibrillar adaptations that may have resulted in no change in calpain activity when a subsequent energy restriction was imposed. Additionally, the ability to find a statistical difference may have been limited by small sample size in both subjects groups and a high variability in the female subject group (resulting from an uncontrolled exercise regimen). Whether changes in calpain activity directly affect changes in muscle function was not demonstrated. This also may have required a more severe and lengthy energy restriction. Both of these associations, energy restriction on substrate oxidation, and calpain activity on muscle function, have meaningful nutritional and functional implications. Energy restriction induces changes in substrate oxidation. Although the results between substrate oxidation and calpain activity are inconclusive, changes in substrate oxidation may induce an increase in myofibrillar calpain activity. Further research is required to establish this in human subjects. Furthermore, the relationship between calpain activity and muscle function is inconclusive and may depend on gender and training status. Definition of this relationship may be important because an increase in calpain activity may result in a decrease in muscle function which can affect performance of individuals in occupations involving physical labor, as well as performance of athletes in both recreational and competitive sports.  151  REFERENCES Acheson K.J., Schutz Y., Bessard T., Ravussin E., Jequier E., Flatt J.P.. Nutritional influences of lipogenesis and thermogenesis after a carbohydrate meal. Am. J. Physiol.. 246:E62-70, 1984. Alexander M.J.L.. 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Wing S.S., Banville D „ 14 kDa ubiquitin-conjugating enzyme structure of the rat gene and regulation upon fasting and by insulin. Am. J. Physiol.. 267: E39-E48, 1994. 162  REFERENCES  Wing S.S., Goldberg A.L.. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting. Am. J. Physiol.. 264: E668-E676, 1993. Wolfe R.R., Klein S., Carraro F., Weber J.M.. Role of triglyceride-fatty acid cycle in controlling fat metabolism in humans during and after exercise. Am. J. Physiol.. 258:E383-E389, 1990. Wong T.S., Booth F.W.. Protein metabolism in rat gastrocnemius muscle after stimulated chronic concentric exercise. J. Appl. Physiol.. 69(5): 1709-1717, 1990. Young D.R., Shapira J., Forrest R., Adachi R.R., Pelligra R.. Model for evaluation of fatty acid metabolism for man during prolonged exercise. J. Appl. Physiol. 23: 716, 1967. Zaidi S.I.M., Narahara H.T.. Degradation of skeletal muscle plasma membrane proteins by calpain. J. Membrane Biol.. 110:209-216, 1989.  163  Appendix 1  Three-day rotating menu plan for a caloric intake of 2700 K c a l  Day 1  Breakfast Amt Type  Lunch Amt Type  Dinner Amt Type  238 ml 22 g 88 g  222 g 14 g 130 g 10 g 100 g 70 g 250 ml  60 g 88 g  7g 20 g 25 g 238 ml Day 2  250 ml 30 g 88g  7g 20 g 25 g 250 ml Day 3  250 ml 26 g 88 g  7g 20 g 25 g 250 ml  orange juice Cheerios Whole Wheat Bread Margarine Peanut Butter Jam Skim milk  Apple juice 145 Shreddies g Whole 21 g Wheat 250 Bread g Margarine 28 g Peanut Butter 166 Jam g Skim M i l k  Orange Juice Raisin Bran Whole Wheat Bread Margarine Peanut Butter Jam Skim M i l k  350 g 80 g 30 g  Spaghetti Margarine Tomato Sauce Parmesan Banana Ice cream Skim milk  14 g 20 g 166g 15 g  Mixed vegetables Margarine Rice  60 g 50 g 88 g  Shortbread Cookies Apple  14 g 20 g 120 g 200 g  Meat Lasagna Grapes Chocolate chip cookies  80 g 88 g  14 g 20 g 160 g 55 g 100 g  164  Amt  Snack Type  Cheese Whole wheat bread Margarine Mayonnaise Apple Salad  89 g 14 g 250 ml  Muffin Margarine Skim milk  Tomato Cucumber Whole wheat bread Margarine Mayonnaise Grapes  89 g 14 g 250 ml  Muffin Margarine Skim milk  89 g 14 g 250 ml  Muffin Margarine Skim milk  Yogurt  Tuna Whole wheat bread Margarine Mayonnaise Yogurt Banana Carrot sticks  Appendix 2 Mean nutrient intake in experimental diets*.  Nutrient  Day 1  Day 2  Day 3  Average  calories (kcal)  2740  2634  2681  2685  Protein (g)  96.7  72.0  126.0  98.2  Carbohydrates (g) Fiber (g)  362.0  416.0  368.0  382.0  31.0  35.9  31.6  32.8  Total Fat (g)  97.5  76.0  81.0  84.8  Saturated Fat (g)  39.3  23.3  33.2  31.9  Monounsaturated Fat(g) Polyunsaturated Fat (g) Cholesterol (mg)  45.1  34.5  39.0  39.5  38.5  31.2  32.2  34.0  166  83.7  156  135  Vitamin A Carotene (RE) Vitamin A Preformed (RE) Vitamin A - Total (RE) Thiamin (mg)  251.0  688  3017  1319  1566.0  990  1142  1233  1817.0  1678  4159  2551  1.84  2.04  2.15  2.01  Riboflavin (mg)  3.03  2.34  2.87  2.74  Niacin (mg)  20.7  20.9  31.8  24.5  Vitamin Bg (mg)  2.61  1.51  2.52  2.22  Vitamin B 1 2 (mg) Folacin (ug)  5.32  2.94  6.46  4.91  315  252  375  314  Pantothenic Acid (mg) Vitamin C (mg)  7.12  6.37  6.26  6.58  179  52  166  132  Vitamin E (mg)  27.5  16.6  26.2  23.4  Calcium (mg)  2007  1287  1917  1737  Copper (mg)  1.77  1.86  2.30  1.98  Iron (mg)  16.0  17.1  29.5  20.9  165  Magnesium (mg)  507  465  504  492  Phosphorus (mg)  2151  1765  2252  2056  Sodium (mg)  4357  2792  4254  3801  Potassium (mg)  3763  3140  4004  3636  Selenium (mg)  178  182  224  195  Zinc (mg)  12  11.1  14.2  12.4  Protein (%)  15  11  19  15  Carbohydrates  53  62  54  56  Fat (%)  32  26  27  28  Polyunsaturated: 1.0 : 1.0 1.3 : 1.0 1.1 : 1.0 : 1.0 saturated fat Sodium Potass1.2 : 1.0 0.9 : 1.0 1.0 : 1.1 : 1.0 ium Calcium:Phos0.9 : 1.0 0.7 : 1.0 0.8 : 0.9 : 1.0 phorus *Determined by Food Processor JJ Nutrient Analysis Software (ESHA Research, Salem prior to the study. No significant differences. Comparison's made by student's unpaired t-test.  166  1.0 1.0 1.0 OR).  Appendix 3  DAILY PHYSICAL ACTIVITY RECORD  A.  Running: 1.  D e s c r i p t i o n of r u n :  2.  Time l e n g t h of r u n :  3.  A p p r o x i m a t e mileage c o v e r e d i n t h e r u n :  . 4. 5.  F r e q u e n c y (# of o c c a s i o n s d u r i n g t h e day) : Intensity: a) Check one of t h e f o l l o w i n g o f f u s i n g t h e c r i t e r i a  l i s t e d i n the instructions: Light: Light:  Medium:  Heavy:  s l i g h t change from normal s t a t e  Medium: some p e r s p i r a t i o n , f a s t e r than n o r m a l b r e a t h i n g Heavy: heavy p e r s p i r a t i o n , heavy b r e a t h i n g  b)  Heart rate values a t :  Beginning:  Middle: of  6. Yes  End:  exercise  Does t h i s r e f l e c t a normal, a v e r a g e r u n f o r you? No  167  Appendix 3 (continued)  a c t i v i t i e s other than running f o l l o w i n g questionnaire d a i l y -  F o r  please  • i n d i c a t e the p h y s i c a l a c t i v i t i e s p a r t i c i p a t e d i n on t h i s day:  FREQUENCY  DURATION 15  15-30  31-60  Walking for Leisure Walking for Exercise Exercise CUsse-S Home Exercises Popular Dance Gardening Bicycling Swimming Tennis Baseball/Softball Racquclball Volleyball Basketball Golf Bowling Ice Hockey Cross Couniry Skiing Ice Skating Oilicrs - Please-specify:  168  fill  out the  i n which  you have  ^ 60+  LIGHT  INTENSITY MEDIUM  0? HEAVY  ^  Appendix 4:  IMTEEBS R E Q U I R E S FOR I AND  M U S C L E . FU1  Male subjects are required to participate in a test period of 1 0 1 2 days where diet and exercise regimens will be imposed for the purpose of looking at the effects of weight loss. Diets will will be provided at approximately 75% of usual caloric intake, and subjects will exercise approximately 30 minutes per day. Subjects are required to eat most meals for 10-12 days at the School of Family and Nutritional Sciences. :  The following test will be completed at the beginning and end of the diet and exercise period: 1. Metabolic Rate 2. Body Composition 3. Muscle Function Subjects should be: - healthy males between 1 8 - 3 5 years - non-smokers - average body weight - sedentary - free from chronic illness What subjects get: - free meals for 10 days  For i n f o r m a t i o n c a l l : 8 2 2 - 6 8 6 9 Investigators: Dr. Linda McCargar, School of Family and Nutritional S c i e n c e s Dr. A n g e l o Belcastro, School of Human Kinetics Please take o n e :  co ro ro  ro  CT) CO CTl CO  CT) CD CD CO  OO  l\0  OO KO  ro  CT)  oo  CO  OO  ro ro  ro ro  CO  OO  OO  ro ro  IV) ro  ro ro  CT) OO  CT) . CD CD CO  CT) CD CD CO  CT) OO CT) CO  CT) 00 CT) CO  CT) CT) CO CO  CD  CD  ro ro  ro ro  CD CD CD CO  CD CD CT)  169  co  oo ro ro  co ro ro  co ro ro  OO  CD  CO CO CD CO  CD  CT) CD CT) CO  oo CD CO  oo O) CO  ro ro  oo ro ro  CD  oo ro rv>  CD  CD CO CT)  oo  oo CD CO  ro ro  co  CO CT) CO  Appendix 5:  EEMALE MET <•  IRTONEIRS EEQOTREID TOE €<Q>MIP(0>SOT<0)H AMuD M U S C L E WVn€tmW STOuDY  Female runners are required to participate in a 14 day period where a dietary regimen will be imposed for the purpose of looking at the effects of dieting in athletes. Diets will be provided at approximately 75% or 100% of usual caloric intake, and subjects will be expected to exercise according to their regular schedule. Subjects are required to eat breakfast and lunch at the School of Family and Nutritional Sciences, as ALL MEALS WILL BE PROVDIED FOR 14 DAYS by the School of Family and Nutritional Sciences. The following tests will be completed in this time period: 1. Metabolic Rate 2. Body Composition 3. Muscle Function (through a muscle biopsy and a Kin-corn test) ©TH&j5©©ii©  sfla®tfflS<S lb©;  - Healthy females between 18-35 years old - Running at least 50 km/week - Average body weight - Free from chronic illness - Taking oral contraceptives What subjects get: - Body composition analysis - Diet analysis - Muscle function and muscle protein analysis .- Free meals for 14 days at For  a 100% or a 75 % maintenance diet  information call:  Investigators:  822-6869  Sab,„a Partes. Schoo! of FamMy and Nutritional Science,  170  Appendix 6  CONSENT FORM  The Investigators:  University of  B r i t i s h Columbia  S.C. Parkes BSc. , Dr. A. B e l c a s t r o ,  T i t l e of the P r o j e c t :  Dr. L. McCargar • Dr S. Barr.  /  Human Muscle F u n c t i o n and S t r u c t u r e F o l l o w i n g a Combined Energy Reduced D i e t and Increased Energy E x p e n d i t u r e  Many people i n Canada are i n v o l v e d i n weight r e d u c t i o n programs, which i n v o l v e e x e r c i s i n g and c a l o r i e r e d u c t i o n . The purpose of t h i s p r o j e c t i s t o determine i f the energy d e f i c i t c r e a t e d by e x e r c i s e and a low c a l o r i e i n t a k e comprimises muscle f u n c t i o n and muscle s t r u c t u r e . will  I understand t h a t as a p a r t i c i p a n t i n t h i s study, I be r e q u i r e d t o :  1.  Keep one 4-day food record, answer q u e s t i o n s my d i e t and l i f e s t y l e .  2.  Have body weight, height, and body composition c a l i p e r s ) measured at the beginning and end of study.  3.  Be w i l l i n g t o f o l l o w a d i e t f o r 10 days which encourages a decreased i n t a k e of c a l o r i e s . This diet w i l l i n c l u d e r e g u l a r foods t h a t . w i l l be p r o v i d e d by the. N u t r i t i o n a l Sciences department at UBC.  4.  Be w i l l i n g t o eat b r e a k f a s t and lunch at UBC, and t o eat d i n n e r and a snack as prepared by a UBC student wherever you f e e l comfortable.  5.  Be w i l l i n g t o p a r t i c i p a t e i n an e x e r c i s e program c o n s i s t i n g of 30 minutes/day f o r a 10 day p e r i o d . 30 minute s e s s i o n w i l l be l e a d by a knowledgable supervisor.  6.  Be w i l l i n g t o come t o UBC measured a t the beginning  7.  Be w i l l i n g t o do a 24 hour u r i n e  8.  Be w i l l i n g t o undergo a muscle biopsy by a t r a i n e d d o c t o r , as w e l l as undergo, a l e g muscle f u n c t i o n t e s t  and and  7  regarding (using the  Each  have my m e t a b o l i c r a t e end of the study. collection.  on a Cybex machine a t the Sports medicine c l i n i c UBC.  at  I t i s u n l i k e l y that any s i d e e f f e c t s w i l l o c c u r as a r e s u l t of p a r t i c i p a t i n g i n t h i s study. My p a r t i c i p a t i o n i n t h i s study i s v o l u n t a r y . I am aware t h a t I may r e f u s e t o p a r t i c i p a t e or t h a t I may withdraw from t h e study a t any time. I f I have f u r t h e r questions c o n c e r n i n g t h i s study, I can c o n t a c t Sabina Prkes a t 224-9175 or Dr. L i n d a McCargar a t 822-6869, or Dr. Angelo B e l o c a s t r o a t 822-3685. Thus, I hereby v o l u m t a r i l y consent t o p a r t i c i p a t e i n t h i s study and acknowledge t h a t I ahve r e c e i v e d a copy of t h i s consent form. I have read and understood the c o n t e n t s of t h i s form. Date: S i g n a t u r e of P a r t i c i p a n t : S i g n a t u r e of Witness:  172  THE UNIVERSITY OF BRITISH COLUMBIA Appendix 7 School of Family and Nutritional Sciences Division of H u m a n Nutrition 2205 East M a l l Vancouver, B . C . Canada V 6 T 1Z4  CONSENT FORM The  U n i v e r s i t y o f B r i t i s h Columbia  Investigators:  S.C. Parkes B S c , Dr. L. McCargar, Dr. A. B e l c a s t r o , Dr. S. B a r r .  T i t l e of the Project:  Human Muscle F u n c t i o n and S t r u c t u r e O c c u r r i n g w i t h a High L e v e l o f Energy Expenditure and a Low Energy I n t a k e  Many female a t h l e t e s t r a i n i n g a t a h i g h l e v e l o f i n t e n s i t y i n Canada have i r r e g u l a r e a t i n g p a t t e r n s , o f which a low energy i n t a k e i s one example. The purpose o f t h i s p r o j e c t i s t o determine i f the energy d e f i c i t c r e a t e d by a h i g h l e v e l o f a c t i v i t y and a low c a l o r i e i n t a k e compromises muscle f u n c t i o n and s t r u c t u r e , and t h e r e f o r e athletic performance. I understand t h a t w i l l be r e q u i r e d t o :  as a p a r t i c i p a n t i n t h i s  study,  1.  Keep a 4-day food  2.  Have my body weight, height, and body composition c a l i p e r s ) measured.  3.  Keep a r e c o r d o f t h e e x e r c i s e performed f o r a 14 day period.  5.  Be w i l l i n g t o come t o UBC and have my m e t a b o l i c measured twice.  6.  Be w i l l i n g t o do two three-day u r i n e  7.  Be w i l l i n g t o undergo a muscle b i o p s y by a t r a i n e d d o c t o r , as w e l l as undergo a l e g muscle f u n c t i o n t e s t on a Kin-corn machine a t the R e h a b i l a t i o n Centre a t UBC.  8.  Be w i l l i n g t o undergo a V 0 max t e s t a t the S p o r t s Medicine C l i n i c a t UBC.  I  record.  2  173  (with  rate  collections.  9.  10.  B e w i l l i n g t o f o l l o w a d i e t f o r 10 d a y s w h i c h encourages a decreased intake/normal intake of c a l o r i e s . This d i e t w i l l include regular foods that w i l l be p r o v i d e d by t h e N u t r i t i o n a l S c i e n c e s D e p a r t m e n t a t UBC.  Be w i l l i n g t o e a t b r e a k f a s t and l u n c h eat d i n n e r and a s n a c k as p r e p a r e d by w h e r e v e r you f e e l c o m f o r t a b l e .  a t UBC, and t o a UBC student  O v e r a l l , t h e s t u d y r e q u i r e s a b o u t 2.5 h o u r s / d a y . This i n c l u d e s time r e q u i r e d f o r i n g e s t i o n of a l l meals f o r the day, r e c o r d i n g e x e r c i s e d o n e t h a t day, and m e a s u r e m e n t s made a t t h e b e g i n n i n g and end o f t h e s t u d y . I t i s u n l i k e l y t h a t any s i d e e f f e c t s w i l l o c c u r a s a r e s u l t o f p a r t i c i p a t i n g i n t h i s s t u d y as an e x p e r i e n c e d p h y s i c i a n w i l l perform the biopsy. However, f o u r m o d e r a t e h e m a t o m a s i n 1000 b i o p s i e s h a v e b e e n r e p o r t e d . The m u s c l e b i o p s y may c a u s e some e a r l y d i s c o m f o r t , s u c h a s m i l d l o c a l t e n d e r n e s s , a n d may c a u s e m i l d t o m o d e r a t e l i m i t a t i o n s i n daily activities. My p a r t i c i p a t i o n i n t h i s s t u d y i s v o l u n t a r y . I a w a r e t h a t I may r e f u s e t o p a r t i c i p a t e o r t h a t I may w i t h d r a w f r o m t h e s t u d y a t any t i m e .  am  The p r i n c i p a l i n v e s t i g a t o r s , t h e 3 c o - i n v e s t i g a t o r s a n d t h e i r a s s i s t a n t s , a n d I w i l l b e t h e o n l y p e o p l e who will have a c c e s s t o the data u n t i l i t i s i n p u b l i s h e d form. As a s u b j e c t , I w i l l h a v e a c o d e n u m b e r b y w h i c h a l l my d a t a and i n f o r m a t i o n w i l l be i d e n t i f i e d . My c o d e n u m b e r w i l l be. k e p t c o n f i d e n t i a l by t h e i n v e s t i g a t o r s .  can at  I f I have f u r t h e r q u e s t i o n s c o n c e r n i n g t h i s s t u d y , I c o n t a c t S a b i n a P a r k e s a t 224-9175 o r D r . L i n d a M c C a r g a r 822-6869, o r D r . A n g e l o B e l c a s t r o a t 822-3685.  Thus, I hereby v o l u n t a r i l y consent t o p a r t i c i p a t e i n t h i s s t u d y and a c k n o w l e d g e t h a t I h a v e r e c e i v e d a copy of t h i s consent form. I have read and understood the contents of t h i s form. Date:  Signature of P a r t i c i p a n t : S i g n a t u r e of Witness: 2 174  Appendix 8  GUIDELINES  FOR  KEEPING A FOOD  RECOUP  A POOD RECORD IS A D E T A I L E D D E S C R I P T I O N OF EACH FOOD OR BEVERAGE ITEM TAKEN OVER 2 4 HRS OF A DAY. AN ACCURATELY COMPLETED FOOD RECORD CAN PROVIDE VALUABLE INFORMATION ABOUT THE NUTRITIONAL CONTENT OF AN I N D I V I D U A L ' S USUAL D l E T TO ASSESS YOUR D I E T RECORD CORRECTLY, WE MUST BE A B L E TO CLEARLY PICTURE THE FOODS AND BEVERAGES T H A T YOU HAVE RECORDED. THE GUIDELINES BELOW D E S C R I B E THE INFORMATION THAT IS IMPORTANT FOR YOU TO RECORD. P L E A S E READ T H E S E G U I D E L I N E S BEFORE YOU START. PLEASE KEEP A RECORD OF EVERYTHING T H A T YOU EAT OR DRINK T H E ATTACHED FORMS FOR 4 DAYS I N A ROW. I N C L U D E 1 WEEKEND DAY THE RECORDING PERIOD.  1.  THE PORTION S I Z E (QUANTITY) NEEDS TO BE ACCURATELY -Please don't guess i f you can measure!  ON IN  RECORDED  It may be h e l p f u l t o m e a s u r e how much y o u r r e g u l a r g l a s s e s , c u p s and b o w l s c o n t a i n b e f o r e y o u s t a r t . You c a n d e s c r i b e p o r t i o n s i z e s i n as many ways as y o u l i k e . The a t t a c h e d f o o d p i c t u r e s a r e p r o v i d e d t o h e l p you w i t h p o r t i o n s i z e s . For  example,  you m i g h t  record:"  Volume 1 c u p or 8 oz or 250 mL o f 2% m i l k 1 t a b l e s p o o n o r 15 mL o r p e a n u t b u t t e r 1 t e a s p o o n or 5 mL o f s u g a r or honey  o r cream  cheese  S ize 1 "2 I n c h by 3/4 i n c h by 3/4 i n c h " 1 medium egg, p o a c h e d smal1 a p p l e 1 2" d i a m e t e r d i g e s t i v e b i s c u i t i medium b r a n m u f f i n  oiece  of cheddar  cheese  We i g h t 2 o u n c e s or 60 grams o f l e a n hamburger (use l a b e l s on p a c k a g e s t o h e l p y o u !  175  m e a t or c h i c k e n o r  fish  TO  HELP  IK  KICS'tiNIilG  OIMU:ilH">  !  176  177  VE1CIITS CONT'D  178  Appendix 9  T I ME & Hour & Min. AM/ PM  PLACE  jFOOD  HOME :  (Use 3 s e p a r a t e line for each J i (. e ro s . D e s c r i b e c a r c f u l l y as 1 i f' wr i t i ri g a r e c i p e :  AWAY  &  8EVERACE  ITEMS  QUANTITY  .  • ' • ••  Specify e a c h Fc me a s'u re : g. .-. ..Us" 9 • Up. cup . G z  -  •  •  Numbe r  179  •£ nl e  Appendix 10  j i  1983 Metropolitan Life Insurance Values for Desirable Weights for Height for AduLts V.-_.(jhts arc lor the lowest mortality in apes 25-Ii? years, Individ u k were with 7.5 o n (I in) heels.  -,-„;l.-d >v.-.,ri,, . indoor cl. r  I  Males 7A. Desirable Weight (kg) Height cms  Small Frame  Medium Frame .  Large Frame  158  S83-61.0  S9.6-642  62.8-68.3  ,  s  wri,..,.,,,,. , . ,  k  f  i Q  |  b  )  .... fi'UAlis Height * cms 148.  .  Small -.Frame 46.4-50.6  Medium Frame  Urge  49.6-55.1  S3.7-S9.8  159  55.6-61.3  S9.9-64.S  63.1- 68.8  160  149  46.6-51.0  59.0-61.7  50.0-SS.5  603-64.9  635-69.4  ISO  46.7-513  S4.1-60.3  161  S03-5S.9  59.3- 6 X 0  60.6- 652  S4.4-60!9  63.8-69.9  162  1S1  46,9-Sl.7  59.7-62.4  50.7-56.4  61.0-65.6  64.2- 70.S  152  S4.8-61.4  163  47.1-521  60.0-62.7  S1.I-S7.0  613-66.0  64.5- 71.1  164  153  SS.2-61.9  47.4-S2.5  SI5-S75  60.4-63.1  61.7-6 6 5  64.9-71.8  55:6-62.4  165  154  47.8-S3.0  51.9-S8.0  60.8- 6 3 5  62.1-67.0  S6.2-63.0  653-725  166  155  48.1-53.6  S2.2-S8.6  61.1-63.8  62.4-675  65.6- 73.2  56.8-63.6  167  156  48.5-54.1  52.7-59.1  61.5-64.2  62.8-68.2  66.0-74.0  157  S73-64.1  168  48.8-54.6  532-59.6  61.8-64.6  632-68.7  66.4- 74.7  S7.8-64.6  169  1S8'  49.3-SS2  62.2- 6S.2  53.8-607  63.8-6 9 3  67.0-75.4  S8.4-6S.3  170  159  49.8-S5.7  543-607  62-5-65.7  643-69.8  67.5- 76.1  53.9-66.0  171  160  503-55 2 .  62.9-66.2  S4.9-612  64.9- 703  68.0-76.2 .  S9.4-66.7  172  161  50 8-56.7  55.4-61.7  63.2-66.7  65.4-70.8  63.9-775  59.9-67.4  173  162  51 4-57.3  63.6-67.3  55.9-623  65.9-71.4  69.1.782  60.5-68.1  174  163  51.9 57 8  63.9-67.8  56.4-63 3  66.4- 71.9  69.5-78.9  61.0-68.8  175  164  52 S-58.4  64.3- 6 8 3  S7.0-63.4  66.9-72.4  70.1.79.5  61.5-695  176  165  53.0-58.9  64.7-68.9  5 7 . 5 « 9  67.5- 73.0  62.0-70.2  70.7- 80.3  177  146  65.0- 69.5  53.6-59.5  58.1-64.5  68.1- 73.5  62.6-70.9  71.3- 81.0  178  167  54.1-600  65.4- 70.0  SS.7-65 0  63.2-71.7  69.6- 74.0  71.9-81.2  179  165  65.7-70.5 "  54.6-60.5  S92 -6S.5  69.2-74.6  63.7-72.4  723-82.5  180  i69  66.1- 71.1  55.2-61.1  59.7-65.1  69.7- 75.1  643-73.1  181  72.8- 83.3  170  66.6- 71.6  55 7-61.6  60.2-66.6  702-7S.S  64.8-73.8  182  73.4- 84.0  171  67.1- 72.1  562-62.1  60.7-67.1  70.7-76.5  6S.3-74.5  183  73.9- 84.7  :72  67.7- 72.7  56 8-62 6  61.0-67.6  71.3- 772  6S.8-7S 2  184  74.5- 85.4  173  68.2- 73.4  573-63.2  61.8-682  71.6- 77.9  66.4-75.9  ISS  75.2-86 1  174  63.7- 74.1  57.6-63.7  62.3-68.7  72.4- 78.6  66.9-76 4  75.9-86.3  186  175  69.2- 74.8  583-64.2  C2.8-69 2  73.0- 793  67.4-76.9  187  76.6- 87.6  176  69.8- 7S.5  58.9-64.8  63.4-69 S  73.7-80.0  773-88.5  68.0-77.5  188  177  70.3- 76.2  59.5-65.4  64.0-70.4  74.4-80.7  65.5-78.1  78.0- 89.4  189  17B  70.9- 76.9  60.0-65.9  74.9-81.5  64.5-70.9  69.0-78.6  190  78.7- 903  17?  71.4- 77.6  7S.4-S22  60.5-66.4  65.1 -71.4  69.5-79.1  191  79.4-91.2  72.1-78.4  ISO  61 0-66.9  65.6-71.9  76.1- S 3 0  803-92.1  70.1-795  192  72.8-79.1  16!  61 6-67 5  66.1-72 5  76.8-83.9  812-93.0  70.7-80.2  193  IS."  73.5- 79.8  62.I-6S.0  77.6-84.8  66.6-7 3 0  71.2-80.7  82.1- 93.9  7 B . Desirable Weipht (lbs) Height ft inch  Small Frame  Medium Frame  5  2  Large Frame  Height fl l.nch  128-134  SmaU Frame  131-141  Medium Frame  Large Frame  5  138-150  4 lu  3  130-136  102-111  133-143  109-121  P.S-131  5 <  140-153  4 !1  132-138  103-113  135-145  142- 1S6  110-123  120-134  5  5  134-140  5  0  • 04-115  137-KS  113-126  122-137  5  6  144-160  5 •  136-142  106-116  139151  115-129 -  125-140  5 7  146-164  138-145  142-154  s :  108-121  118-132  126143  S 8  149-16S  140-146  145-137  > 1 1124  •21-135  13 i -147  152^ 172  5  9  142-151  5 4  114-127  148-160  124-138  134-151  5 10  155-176  4  144-154  • 17-130  151-163  137-155  5  11 .  158-150  127-141  146-1S7  5  154-166  120-133  140-159  161-184  130-144  6  0  149-160  5 7  123-136  145-163  157-i 70  133-147  6 1  164-16S  152-164  5 S  126-139  165-192  • 36-150  146-167  160-174  6 2  IS5-I6S  5 S-  164-178  3  ISS-172  167-1 S3  132-14:.  142-156  157-173  176-202  \i  13?-] S3  149-170  17219?  129-142  6 4  162-176  17I-IS7  IS 1-207  6  S o u r c e . K e f 2 , i ; . S 3 . v a l u e s ^ r c b a s e d o n ri^t? M e d i c a l Directors of Americ.i  5  5 5  e o , k - c : e d in i h c ) 9 ? V b u . J .  :  ~ -  !35-14->: .  (. '  vud  180  y .1!  159  1 S5.-176 _i56-J.7?_.  • HS-lfr? "V ••>•:  r,.^ i . . .  :  U / e Ir-sura:-  Anthropometric Measurement Standards  Looking straight ahead with Frankfurt plane horizontal  3  Frankfurt plane  Shoulders relaxed Shoulder blades, buttocks, and heels touching measurement board  Arms at sides  Legs siraight and knees together  Feet flat and wilh heels almost together  for P.anning [ntervencion b y M D  S.mko  181  Guide  Subscapular Skinfold Recommended Technique The subscapular skinfold is picked up on a diagonal, inclined infero-laterally approximately 45° to the horizontal plane in the natural cleavage lines of the skin. The site is just inferior to the inferior angle of the scapula (see Figure 3). The sub-  Figure 3 Landmarks for subscapular and triceps skinfolds.  ject stands comfortably erect, with the upper extremities relaxed at the sides of the body. To locate the site, the measurer palpates the scapula, running the fingers inferiorly and laterally, along its vertebral border until the inferior angle is identified. For some subjects, especially the obese, gen. tie placement of the subject's arm behind the back aids in identifying the site. The caliper jaws are applied 1 cm infero-lateral to the thumb and finger raising the fold, and the thickness is recorded to the nearest 0.1 cm (see Figure 4).  182  0.88 (Sloan & Shapiro, 1972) to 1.53 mm (Johnston et al., 1972). Sources of Reference Data Children  Johnston et aL, 1972, 1974 Adults  Durnin & Womersley, 1974 Stoudt et al., 1970 Figure 4 Measurement of subscapular skinfold.  Midaxillary Skinfold  Purpose  Recommended Technique  Subscapular skinfold thickness is a measure of subcutaneous adipose tissue and skin thickness on the posterior aspect of the torso. It is an important measure of nutritional status and, in combination with other skinfold measurements, is a useful predictor of total body fat, blood pressure, and blood lipids.  Midaxillary skinfold thickness is measured at the level of the xiphi-sternal junction, in the midaxillary line, with the skinfold horizontal (see Figure 5). The subject stands erect, except that young in-  Literature  The International Biological Programme recommended the subscapular skinfold thickness as one of 21 basic measurements to be included in survey studies of growth and physique (Weiner & Lourie, 1981). Cameron (1978), citing the work of Dumin and associates (Dumin & Rahaman, 1967; Durnin & Womersley, 1974) recommended the subscapular skinfold thickness, in combination with the triceps, biceps, and suprailiac skinfold thicknesses,as the smallest number of skinfolds representative of body fat. Together with the triceps skinfold thickness, this site is used in health-related fitness tests for children. There has been general agreement on the location of the subscapular skinfold site, although some authors recommend measuring a verficular skinfold (Cameron, 1978). A diagonal fold, in the natural cleavage of the skin, at the inferior angle of. the scapula, is recommended because this makes it easier to raise a fold. Reliability  The reproducibility of the subscapular skinfold measurement is good. Inrrameasurer errors range from 0.88 (Lohman, 1981) to 1.16 mm (Wilmore & Behnke, 1969). Intermeasurer errors range from  183  Figure S illustration of the level of the xiphi-stemal junction at which the midaxUlary skinfold is measured.  fants sit on the lap of the mother or caretaker. Care is taken to ensure that the subject does not flex the trunk towards the side being measured. The left arm is slightly abducted and flexed at the shoulder joint (see Figure 6). A bra can be worn while the measurement is made, but the strap may have to be undone. The measurer stands facing the side of the subject to be measured, elevates a horizontal skinfold with the left hand, and measures its thickness to the nearest 0.1 cm (see Figure 7). Purpose Midaxillajry skinfold thickness is a guide to the total amount and the distribution of trunk subcutaneous adipose tissue. It is less highly associated than  level usually corresponds to the fifth rib in the midaxillary line except in women. The midaxillary skinfold, as the name implies, is measured in the midaxillary line (Johnston et al.. 1974. Pascale et al.. 1956; Young. 1464), but Parizkova (1961) measured in the anterior axillary line. . . . The fold should be parallel to the cleavage lines of the skin at the site. These lines are nearly horizontal in the midaxillary line at the level of the xiphoid process. Nevertheless, this measurement was made across a vertical fold by Wilmore and Behnke (1969). Pascale et al (1956) measured a vertical fold unless "the lines of Langer resulted in tension of the skinfold. Then the skinfold was taken along these lines." A fold at 45° to the horizontal was measured by Slaughter et al. (1978). Differences in thickness between horizontal and vertical folds are minimal (Chumlea & Roche, 1986). Reliability  Figure 7  Measurement of midaxillary skinfold.  the subscapular skinfold thickness with the total trunk adipose tissue. It is easier to measure the midaxillary skinfold than the subscapular skinfold in bedfast individuals, and the former skin/old is less likely to be affected by edema. Also, the midaxillary skinfold is easier to measure than most other trunk skinfolds in the obese because it tends to be thinner (Johnston et al., 1974).  Using the value SD of difference/ J2. the interobserver reliability was 1.47 mm in children aged 6 to 11 years in a National Center for Health Statistics Survey (Johnston et al., 1972) and was 0.36 mm for children and 0.64 mm for adults in the Fels Longitudinal Study (Chumlea & Roche, 1979). The intraobserver reliability has been reported also as the SE from a regression/v'2 with values of about 1.0 mm (Wilmore & Behnke, 1969). Zavaleta and Malina (1982) reported a technical error of 0.95 mm for Mexican-American boys and of 2.08 mm for Health Examination Survey data. Lohman (1981) estimated the intrameasurcr eircr as 1.22 mm. Sources of Reference Data  Literature  Children  The literature includes few descriptions of the positioning of a subject for the measurement of the midaxillary skinfold, but the positioning in the recommended technique is in agreement with the usual practice Most have related the level of measurement to a bony landmark, most often the xiphoid process (Oberman et al., 1965; Pascale et al., 1956; Young. 1964). Less commonly, the measurement has been made at the level of the fifth rib (Slaughter et al., 1978; Wilmore & Behnke, 1969), or the ninth or tenth rib (Lohman et a!., 1975). lt has been measured midway between the nipples and the umbilicus (Johnston et al., 1974). and at the level of the nipples (Johnston et al., 1972). The latter  184  Johnston et al., 1972. 1974 Adults  Oberman et al.. 1965 Young, 1964  Pectoral (Chest) S k i n f o l d Recommended Technique It is recommended that the same pectoral (chest) skinfold site be used for both males and females (see Figure 8). Pectoral skinfold thickness is measured using a skinfold with its long axis directed  relaxed at the sides. For a patient confined to a wheelchair, the measurement can be made with the subject in a wheelchair with the arms relaxed at the sides.  Purpose  Pectoral skinfold thicknesses have high correlations with body density determined by hydrostatic weighing (Pollock et al., 1975,1976). This measure has been selected by regression analysis for inclusion in equations to predict body density from anthropometric values (Pascale et al., 1956).  Figure 8 Illustration of location of pectoral skinfold in males and females.  to the nipple. The skinfold is picked up on the anterior axillary fold as high as possible; the thickness is measured 1 cm inferior to this (see Figure 9). The measurement is made to the nearest 0.1 cm while the subject stands with the arms hanging relaxed at the sides (see Figure 10). For a patient confined to bed, the measurement is made while the patient is supine, with arms  Figure 9 Location of pectoral skinfold on the anterior axillary fold.  Figure 10 Measurement of the pectoral skinfold.  Literature Pectoral skinfold thicknesses are not measured commonly. Its exclusion from many studies may result from the vague descriptions in the literature and the complexity o f the measurement. Complications include the need for the removal of a Tshirt or undergarment a n d the need to measure away from the mammary gland in women. In most studies, a distinction is not made between the sexes in the methods for the measurement of pectoral skinfold thicknesses. Three sites for the measurement of pectoral skinfold thicknesses are described: (a) the midpoint between the anterior axillary fold a n d the nipple (Pascale et al., 1956; Pollock et al., 1980); (b) juxtanipple (Pascale et a l . , 1956; Forsyth & Sinning, 1973); and (c) medial to the anterior axillary fold (Katch & Michael, 1968). Skerlj et al. (1953) appear to describe a pectoral skinfold site located between those described i n m e t h o d s (b) a n d (c) above. Hertzberg et a l . (1963) describe the pectoral skinfold site as juxtanipple, but their illustration shows the site as described under (a) above. The literature does not indicate the direction from the nipple of the juxtanipple s k i n f o l d site. Skerlj et al. (1953) describe the site as being at the axillary border of the pectoralis major muscle and state that the location is somewhat more proximal for w o m e n than for m e n . Pollock et al. (1984) describe the pectoral skinfold site as (a) above for males and used o n e third o f the distance between the anterior axillary fold a n d the nipple for women. Depending o n the size of of the mammary gland, this description for w o m e n w o u l d lead to variable site location. T h e m a i n intent of describing a separate site for females was to keep the measurement away from the g l a n d u l a r tissue of the mammary gland.  185  The literature is vague, but the general impression is that most investigators measured the thickness of an oblique fold along the line of the anterior axillary fold. Hertzberg et al. (1963) measured the thickness of a vertical fold. The same site for both males and females is desirable. Although the midpoint between the anterior axillary fold and the nipple is used most commonly for males, it is not appropriate for females. Because of the variability in the size of the mammary gland, it is difficult to use the nipple as a reference point to locate the site. Also, in most cases it would be difficult to exclude mammary tissue from measurements at the site described under (a). The recommended site allows the measurement to be made while a woman wears a two-piece bathing suit or bra.  dominal wall with normal respiration. The subject stands erect with body weight evenly distributed on both feet. Children stand on a platform to allow the measurer appropriate access to the skinfold site. Select a site 3 cm lateral to the midpoint of the umbilicus and 1 cm inferior to if (see Figure 11). The decision whether to measure to the left or right of the umbilicus should be consistent within a study. Raise a horizontal skinfold with the left hand and measure its thickness to the nearest 0.1 cm (see Figure 12).  Reliability tntrameasurer reliability coefficients are very high, ranging from .91 to .97 (Pollock et al., 1975. 1976). The standard error of measurement (SEM) generally averages 1 to 2 mm. Data from 68 adults showed a correlation of .96 between trials measured on separate days with a SEM of 1.45 m m (Pollock, unpublished data, 1985). [ntermeasurer correlations are generally above .9, but the S E M may vary as much as 3 to 5 mm with inexperienced measurers, or when the site is not standardized (Lohman et a)., 1984). Jackson et al. (1978) reported a correlation among measurers of .98 with a SEM of 2.1 mm. An intermeasurer correlation of .93 with a S E M of 1.7 m m has been recorded (Pollock, unpublished data, 1985). Sources of Reference Data Children  none reported Figure 12  Adults  Measurement of abdominal  skinfold.  none reported Purpose  Abdominal Skinfold Recommended Technique For the measurement of abdominal skinfold thickness, the subject relaxes the abdominal wall musculature as much as possible during the procedure and breathes normally. The subject may be asked to hold his or her breath near the- end of expiration if there is bothersome movement of the ab-  186  The abdominal skinfold is measured commonly and has been included in many studies of body fatness and in many regression equations (Lohman, 1981). Abdominal skinfold thickness changes markedly with weight reduction (Despres et al., 1985). It is relatively easy to access, is relatively large, differs considerably among subjects, and is reasonably reproducible with the recommended technique.  Literature Several locations have been used for measurement of the abdominal skinfold. These include adjacent to the umbilicus; level of the umbilicus but 5 cm to the left of it; slightly inferior to the umbilicus and 1 cm to the right of it, and a quarter of the distance between the umbilicus and the anterior superior iliac spine (Edwards, 1950; Lohman, 1981; Parizkovi & Zdenek, 1972; Skerlj et al., 1953; Weiner & Lourie, 1981). Most have measured horizontal fold (Behnke & Wilmore, 1974), but others have measured a vertical fold (Sinning et al., 1985; Steinkamp et al., 1965). Some subjects have a "crease" in the region of the umbilicus that precludes selection of a single site for all, and in the obese it is difficult to raise a discrete skinfold.  Suprailiac Skinfold  Mid-Axillary Line  Figure 13 Diagram to illustrate the location of the suprailiac skinfold in the midaxillary line superior to the iliac crest.  Reliability Wilmore and Behnke (1969) reported a test-retest correlation of .979 for measurements made 1 day apart in young men. An intrameasurer technical error of 0.89 mm was reported by Zavaleta and Malina (1982). Sources of Reference Data Children  None reported Adults  None reported  Suprailiac Skinfold  Figure 1 4 Subject position for measurement of the suprailiac skinfold.  Recommended Technique The suprailiac skinfold is measured in the midaxillary line immediately superior to the iliac crest (see Figure 13). The subject stands with feet together and in an erect position. The arms hang by the sides or, if necessary, they can be abducted slightly to improve access to the site (see Figure 14). In those unable to stand, the measurement can be made with the subject supine. An oblique skinfold is grasped just posterior to the midaxillary line following the natural cleavage lines of the skin. It is aligned inferomedially at 45° to the horizontal (see Figure 14). The caliper jaws are applied about 1 cm from the fingers holding the skinfold, and the thickness is recorded to the nearest 0.1 cm (see Figure 15).  187  Purpose Suprailiac skinfold thicknesses are commonly used as indices of body fatness together with other skinfold thicknesses (Dumin & Womersley, 1974). Suprailiac skinfold thicknesses are useful in the study of subcutaneous adipose tissue distribution, which is important in regard to risk of disease (Lapidus et al., 1984; Larsson et al., 1984).  Literature In most studies, the subjects stood for the measurement of suprailiac skinfold thicknesses. Considerable variation regarding the location and direction of the suprailiac skinfold occurs in the  proach to skinfold measurement of this manual. The use of a vertical fold (Behnke & Wilmore, 1974). horizontal fold (Johnston et al., 1974), or oblique folds at more anterior locations (Pollock et al., 1975; Ross and Maffell-Jones, 1983) is common (see Figure 16). The recommended site of measurement is very, similar to the site sometimes described for the waist skinfold (Behnke & Wilmore, 1974; Brown & Jones, 1977; Skerlj et al., 1953). Because of this similarity, the waist skinfold procedure is not described separately. Reliability  Figure 15  Measurement of the suprailiac skinfold  literature. Thicknesses at the various locations appear highly correlated with each other and with body density (Sinning & Wilson. 1984), so that no one position appears to offer unique information. Relatively large systematic differences in thicknesses among locations emphasize the need to standardize the technique for the measurement of the suprailiac skinfold. The selection of a site on the midaxillary line superior to the iliac creast has the advantage of being easily located in reference to anatomical landmarks. The direction of the fold parallel to the cleavage lines of the skin matches the general ap-  lot  Wilmore and Behnke (1969) reported a test-retest correlation of .970 for values recorded 1 day apart in young men. Technical errors of 1.53 mm in children and youth (Johnston et al., 1974) and of 1.7 mm in adults (Haas &: Flegal, 1981) have been reported, [n each study, the errors for suprailiac skinfold thicknesses were larger than those for other skinfold sites. Intrameasurer technical errors of 0.3 to 1.0 mm have been reported by others (Buschang, 1980; Meleski, 1980; Zavaleta & Malina, 1982). Sources of Reference D a t a Children  Baker et al., 1958 Ferris et al., 1979 Johnston et al., 1974 (horizontal fold) Montoye, 1978 Schutte, 1979 Zavaleta, 1976 Adults  Katch & Michael, 1968  Thigh Skinfold Recommended Technique  I. Siondordized Supro<icac Site Z. After Pollock et al 3. ftfier Ross and Morfell-Jones Figure 16 Location of recommended suprailiac site in reference to oilier frequently measured suprailiac sites.  188  The thigh skinfold site is located in the midline of the anterior aspect of the thigh, midway between the inguinal crease and the proximal border of the patella (see Figure 17). The subject flexes the hip to assist location of the inguinal crease. The proximal reference point is on the inguinal crease at the midpoint of the long axis of the thigh. The distal reference point (proximal border of the patella) is located while the knee of the subject is extended.  Inguinal V^Crease  Mid-Thigh Skinfold Proximal J_ h- Border of Patella  hydrostatic weighing (Wilmore & Behnke, 1969, 1970). Thigh skinfold thickness has been selected by regression analysts as one of the skinfold measures included in equations to predict body density from anthropometric values.  Literature A few early studies refer to both anterior and posterior thigh skinfold sites, but most refer only to the anterior site: thus, further discussion will relate to this site only.  Figure 17 Location of the midthigh skinfold site. The thickness of a vertical fold is measured while the subject stands. The body weight is shifted to the other foot while the leg on the side of the measurement is relaxed with the knee slightly flexed and the foot flat on the floor (see Figure 18). If the maintenance of balance is a problem, the subject holds the top of the measurer's shoulder, a counter top, or high-backed chair. For patients confined to a bed or wheelchair, the thigh skintold is measured while the patient is supine The caliper jaws are applied about 1 cm distal to the fingers holding the fold; the thickness of the fold is recorded to the nearest 0.1 cm. Purpose  Although description of the thigh skinfold site appears to be standardized among many studies, considerable variation can be found (Lohman et al., 1984). The most common description of the thigh skinfold site is on the anterior aspect of the thigh, midway between the hip and knee (Wilmore & Behnke, 1969; Zuti & Golding, 1973). Sloan et al. (1962) used the midpoint from the inguinal crease to the proximal margin of the patella. Others give a more general description, such as halfway down the rectus femoris muscle (Young et al., 1962). The investigators mentioned previously described their measurements as being made with the subject in the standing position, leg relaxed. Some measure with the leg flexed 9 0 ° at the knee by placing the foot on a box. This technique is recommended by Ross and Marfell-Jones (1984). All investigators measure thigh skinfold thicknesses with a vertical fold aligned in the long axis of the thigh.  Thigh skinfold thicknesses have moderate to high correlations with body density determined by Reliability  Figure 18 Measurement of midthigh skinfold  Intrameasurer reliability coefficients are very high, ranging from .91 to .98 (Pollock et al., 1976, Wilmore & Behnke, 1969; Zuti & Golding, 1973), although the standard error of measurement (SEM) generally averages between 1 to 2 mm. Recent data on 68 adults showed a correlation of .985 between trials taken on separate days with a SEM of 1.4 mm (Pollock et al., unpublished data, 1985). Others have reported intrameasurer technical errors of 0.5 to 0.7 mm (Meleski, 1980; Zavaleta, 1976). Intermeasurer correlations are generally above .9, but the SEM may be as much as 3 to 4 mm with inexperienced measurers or when the sites are not standardized (Lohman et al., 1984). Jackson et al. (1978) reported a correlation among measurers of .97 and a SEM of 2.4 mm for measurers of varying experience who had trained together. In an unpublished study. Pollock (1986) showed an intermeasurer correlation of .975, with a SE.M of 2.1 mm.  189  Triceps S k i n f o l d Recommended Technique I'he triceps skinfold is measured in the midline .»l the posterior aspect of the arm. over the triceps muscle, at a point midway between the lateral projection of the acromion process of the scapula and (he interior margin of the olecranon process of the ulna. The level of measurement is determined by measuring the distance between the lateral projection of the acromial process and the interior border of the olecranon process oi the ulna, using a tape measure, with the elbow flexed to K) (see Figure 24). The tape is placed with i'.^ zero mark on the acromion and stretched aW>ne. she upper arm. extending below the elbow The m:ot>-Mnl l  i>  m.irked - n  I!H M l e r d l  side ot liu' .urn '  !*he * - i m . ' . i s u t v d Miv.ndmc. e v . e p i it-: iu'..iniN \\\<- h.i.uheopped. Viw- skml«>id i> nit.-suivd I'M- .\:ni hJH^ing lo<>r-eb .>nd lOniUw:cibW M iS- <i:PK-"i'* side (Pijzure 2v . The c.ilipor is lu'id i " . ; h » ' relit bond. The m e ^ u r e r --UiKibehind i h « . 'liiM^'i .md places the p . i b v i «•»: his c-r her k-ti h.:.id on -he subject's .srm pr^viin.ii to 11Kmarked iv*. o'. v- ;-h ;ho thumb <ind mdov finger directed ;:-«;en.i::v I"he trtcep> >kjn{---id J< picked up \vi(]-» ::v. k-i'l :humh and indov im^'r anpiov in.iU'iv ! ov. pio\;mal to ;he .narked level. .;nd th..tips o: {IK caliper*, .ire applied to t h e skinfold .v. the m.i.'r.vd lev -.•! <<ee I'igure JN i ' h e -site nn'.i^u:*'.';r---ii i v tn the r.iuthi>i-^e'.ii\r,\ ,  l-iyurc 24 I ..ifjt ion of" ir.ui-.ir.r. i.-x. I u; •skinluui  :  190  :  when i!i«- I'.ihn is directed anienorh I'.u hculai problems u ill K- I,iced when nirasurino. the obese .iiul iiHi-ail.i!' subjects with little l.n .il this sit,-. II neceSs.iiA :it Ilu- case ol olvso suh|ecls. .in J>MS t.mi in.iv pi. k up tlu- fold v\ ilh two hands, but ilus i;ives larger readings ih.ui il m i l ' hand is used (Damon. Purpose  Hiceps S k i n f o l d Kecdititnended  Iechnii|ue  Itice:-- vkuiliilJ Ihii kne-s i - measured .is the thickness a vertical lold raised or. tin- anterior aspect < -Ill:-.- a rin. over I he K-Hv o i ilu- biceps muscle (see l-is;i;!i- 27). The skmlold is raised 1 cm superior lo the line marked lor Ilu- measurement of triceps  The triceps skinlold is measured mure commonly than om other, portly because it is so accessible. It is closely correlated with percentage ol hodv l.il and with total body fat but is less well correlated with blood pressure than are trunk skinfolds. Ii is often included in studies ol lal patternim;. Literature The level of the site is marked with the . i n n lle\ed .it a riy;hi ancje af the elbow, and the skinfold is measured with ihe arm hanging louselv at the side. Positioning is not cruci.il. except Ih.i! the subieci should he relaxed and the palm direcied anteriorly so lh.it the posterior midline can he determined Most measure subjects in a slandini; position, though nonambulatorv patients mav he measured when supine Inlants ma\' be measured Ivim; d o w n , o r heme held on someone's b p . When supine or siiiinv; positions are used, ihe recommended lechnicjuc can stil! be applied wi'.h hisic modification 1  Reliability In general, measurement error increase.- with the J U C ol the sublet"I and with increasine, levels oi famous lntemiea<urer technical errors varv from 0..S to 1 S'.' mm (Johnston et al.. 1974: Johnston & Mack. Inlranieasurer technical errors varv from ti.-l to il S mm (Johnston c-l al . l«7-i. 147V Maluia i Ruschanj;. H$4: Martorell el al.. 197-i  I'l^uro  2~  I .«c.ilinn < i - i - o p -  ski-a!.«:d--::i  skin:..; ' thickness and a m i circumference, on a vertic.:] line joining ihe anierior border ol :he .u ro —. -n and (he center of t i n - antecubital fossa •see ure 28). The subieci stands. I<v:r.i> the tvsvJsure:" -.\ ilh tlie upper cvtromitv relaxed al the s:de. and 1::e palm directed .iniersvh The caliper :a--vs are .-'.---.Mied al the marked love! 'see liu'.:re 2S). The thick r.ess of the skinfold is recorded to :ho nearest J  v  :! 1 , -•  Sources of Reference Data C/n7</rf M irisancho. 1"S1 Johnston, et a! . MS! A tl ul l<  Irisancho.  I')s|  lohiir-ion ol a! . M S I  I iRur.-  191  ? s  M i - . i s , , : , : - . . . .  v  . . , >  .1.,,,..  Purpose The biceps skinfold is a measurer of subcutaneous adipose tissue and skin thickness on the anterior aspect of the arm. In combination with other skinfold measurements, it is a useful predictor of total body fat (Durnin & Womersley, 1974). Together with triceps skinfold thickness it may assist the calculation of the "muscle plus bone" cross-sectional area at this level. It can be useful in the obese, in whom many other skinfolds cannot be measured.  Reliability The standard deviation of differences for repeated measurements of biceps skinfold thicknesses by one investigator was 1.9 mm, and the standard deviation of the differences between three measurers was 1.9 mm (Edwards et al., 1955). Technical errors for intrameasurer differences are about 0.2 to 0.6 mm (Meleski, 1980; Zavaleta, 1976).  Literature Biceps skinfold thickness is not measured commonly. Consequently, the site has been poorly described, and the reproducibility of measurement is not well established. The International Biological Programme included the biceps skinfold thickness as one of 10 possible skinfold thickness measurements for use in studies of growth and physique, nutritional status, and work capacity (Weiner & Lourie, 1981). More recently, Cameron (1978). based presumably on the work of Durnin and associates (Durnin & Rahaman, 1967; Durnin & Womersley, 1974), recommended biceps skinfold thickness in combination with the triceps, subscapular, and suprailiac skinfold thicknesses, as the fewest skinfold thicknesses representative of body fat; this recommendation is unlikely to be valid in both sexes and in different age groups. The biceps skinfold thickness is used mainly by workers who employ the Durnin equations to estimate percent body fat or study the obese. Previous descriptions of the biceps skinfold have placed the site generally at the position recommended here. Subject positioning has varied between studies from sitting, with the upper extremity resting supinated on the subject's thigh (Durnin & Rahaman, 1967), to standing with the upper extremity held relaxed at the side, palm facing forwards (Cameron, 1978), as is recommended. This brings (he site into the anterior midline of the arm, and muscle contraction and skin tension are low.  Sources of Reference Data Children  Harsha et al.. 1978 McCowan et al.. 1975 Adults Durnin fc Rahaman, 1967 Durnin & Womersley, 1974 Edwards el al., 1955  192  Appendix 12  The e q u i v a l e n t f a t content, as a percentage o f bodyweight, f o r a range o f values f o r t h e sum o f f o u r s k i n f o l d s ( b i c e p s , t r i c e p s , subscapular and s u p r a - i l i a c ) o f males:  M a l e s (age in years) k1nfolds (mm) IS 20 *5 3° 35 40 45 5° 55 60 65 7° 75 Hz 05 <IO  ''5 100 105 1 10 i 1 5 1 20 1 25 1 30 '35 1 40 '4 5 1 50 55 1  l('C  16? •170 1 7 t iSc >*5 1 yc •95 200 205 2 10  F c m n l c s (net in \o.irs) -  17-29 48 81 105 129 >4'7 164 '77 190 20- I 2 12 222  30-39 — 122 •4-2 l6 2 177 19 2 204 215 22 5  2 R  23 5 24 251 25 9 26 6  5' 5 26 2 26 <> -  272 27R 284  27 6 28 2  290 29 6 30 1 30C1 311 3' 5 3'9 3^3 32'7 331 33'5 33 9 343 34-6 348 — — — — — — —  23 I 2 4 O 4  2  28 8 2() 4 300 30 5 3 1 0 3' 5 320 3 5 32 9 33 3 33 7 34 1 34 5 34 9 3 5:. 356 3 5'9 — — — 2  3  40-49  50 +  1 d-29  30-39  40-49  — I 22 ISO  —  10 s 141 id S  —  —  17-0  19-8 222  •7'7 196 214 230 246 25-9 271 28 2 29-3 303 312 32 ' 33° 33 7 34'4 35 1 358 3(1-4 37 0 37'. 38-2 3R 7 39 2 39/ 402 40 7 412 41 6 420 — — — — — — —  1 26 1 56 1 8-0 208 22 s> 24-7 2O 5 279 29 2 30 4 31 (> 32 7 3- s X  34 3 5  H  3(. 0 374 38 2 3') 0 39-7 40 4 4 1 1  4  S  1  42 4  430 43 <• 44  !  44  (.  45 4S 6 46 1 — 1  — — — — —  19 5 2 i'S 23 4 25-0 z6i 27 8 291 30 2 3 2 51  2  3:> • ;,4 0 34 K .- - (• 3'- 4 .- 7 37 * 3* 4 39 3<i (. 40 2 408 4 1 1 418 42 3 42 8 1  0  433 43 7 - 44 I — — — — — — —  194 218 237 25-5 2(1 9 28 2 29 4 306 3,6 325 334 34 3 ~ : •1  35 X 30 5 37-2 37"'/ 3K 0 1,1 1 3.1 f. 4 C  1  40 6 41 1 4 1 u 42 1 42  0  43 ' 436 44 0 44'4 44 8 •15  -  4 S  6  459 462 46- 5 • —  24 5 26 4 28 2 29 6 31 c 32  1  33 2 34 1 3to 35 9 \i- 7 37 5 tS 3 i'l 0 3'; 7 40 4 4 I O 5 0 425 4 3 0 1  4  4 ;  435 44 c 5  44 4  t  O  5 4 K <0-2  4  4 5  (.  4'.  4  7  C  7-1 -7 X  4  <>- 2 4 ^ S 48S 4-1  1  S° +  _ 214 24 -o 2C 6 28 5 30 3 31 y 33 4 34 357 36-7 37 7 3-7 39 '6 40 4 4 1 "2 419 42O 4 3'3 4 3"'J 445 45 1 45 7 4 0 2 40 7 47 2 47 7 4* 2 4* 7 49 2 <•;•£•  so 0 5° 4 C O k tl 2 51 (. 520 524 52 7  4"l 4 530 I n t uo-thirds of the inst-inccs the error was within j ; 3 5°,„ of i l i t body-weight • S f.:l for tl c l u i n u n and ± 5",. for the men.  •From Durnin and Womersley, 1974.  193  Appendix 13: Anthropometric recording form for male subjects  Age: Name: Date:  .  —  WEIGHT: _ 3  Trials:  K <  HEIGHT:  c  BMI =  m  __kg/m  2  2  SKINFOLDS: Tricep:  1 .  Bicep:  .  Sub-scapular : Suprailiac:  3  — —.  —  .  Sum o f S k i n f o l d s : _ _ % Body F a t :  194  Appendix 14: Anthropometric recording form for female subjects  Name:  Age:  Date:  WEIGHT Trials:  kg  HEIGHT:.  BMI =  kg  k  S  cm  kg/m  ;  2  SKINFOLDS:  1  2  3  Tricep Subscapular Suprailiac Abdominal Chest Axilla Front Thigh  Sum of Skinfolds:  mm  % Body fat:  195  AVERAGE  Appendix 15:  Maximum heart rate, heart rate training range and regression equation (used to determine oxygen inspired/min) for each male subject:  Subject Code  Maximum Heart Rate (beats/min)  Training Heart Rate Range (beats/min)  Regression equation  1  170  102-136  y = 45.9x - 4382.9  2  156  94-125  y = 31.0x - 1822.3  3  183  110-146  y = 28.4x - 1602.5  4  187  112-150  y = 28.2x - 2515.6  5  198  119-158  Not applicable§  6  205  123-164  y = 38.8x - 3940.4  7  189  113-151  y = 24.6x - 1673.3  8 210 126-168 y = 28.8x - 2757.7 *y = Volume of oxygen inspired, ml/min.; x = average heart rate value (beats/min). §Heart rate values were not available for this subject  196  Appendix 16  1 9 7  Appendix 17: Kin-com data collection sheet for male and female subjects  Name:  D a t e :  Age:  -  Body weight:  Leg tested:  EXERCISE STIMULUS Lever arm:  Reps:  Position:  ROM:  Exercise tests: 30/90/120/180 deg/sec, concentric and eccentric knee extension, 4 reps Date  File Name  Velocity  Torque  Eccentric 30 90 120 180  198  Concentric  !  Appendix 18  The University of British Columbia Office of Research Services Clinical Screening Committee for Research Involving Human Subjects  Certificate of Approval PRINCIPAL INVESTIGATOR  DEPARTMENT  McCargar, L J .  Family & Nutr Sci  C94-0025  NSTTTUTlONtS) W H E R E R E S E A R C H WILL B E CARRIED OUT  U B C Campus CO-MVtSDGAlORS  ~ —  —  Barr, S.I., Family & Nutr Sci; Belcastro, AN., Human Kinetics; Parkes, S.C., Family & Nutr SPONSORING AGENCIES  Human muscle function and structure following a low energy intake plus a high activity level APPROVAL DATE  TERM (YEARS)  MODIFICATION OF:  3 CERTIFICATION.  The protocol and consent form for the above-named project have been reviewed by the Committee and the experimental procedures were found to be acceptable on ethical grounds for research involving human subjects.  Dr B^McGillivray, Chair or Dr. A. Hannam, Associate Chair  Jfo £ D.Spratley 7^ 'Director, Research Services D  r  y  This Certificate of Approval is valid for the above term provided there is no change in the experimental procedures  199  Appendix 19  Information regarding male subjects re: ethnic group, employment, and previous activities. ID#  Ethnic Group  Occupation  1  Caucasian  Student/Salesperson  2  Caucasian  Student  Soccer, hockey, weights Running, skiing  3  Caucasian  Student  Hockey  4  Caucasian  Student  Hockey  5  Caucasian  Student  Hockey,weights  6  Caucasian  Student  Hockey, weights  7  Caucasian  Student  None  8  Caucasian  Student  Hockey, skiing, weights  200  Previous  Activities  Appendix 20  EQUATIONS FOR BODY WEIGHT CALCULATIONS 1.  Percentage i d e a l body wight =  2.  Percentage weight l o s s =  a c t u a l weight u s u a l weight  (usual weight - a c t u a l weight) u s u a l weight  201  X 100  Appendix 21 Information regarding female subjects re: ethnic group, employment, type of exercise, etc.. ID #  Ethnic Group  Occupation  Type of Exercise  Years involved in activities  1  Caucasian  Student  16  2  Caucasian  Student  Run, Bike, Swim, Weights (Wts) Run, Bike, Wts  3  Caucasian  Student  Run, Bike, Walk, Field Hockey  9  4  Caucasian  Student  5  Caucasian  6  Caucasian  Student  Run, Bike, Swim  5  7  Caucasian  Student  Run, Bike, Wts, Karate  16  8  Caucasian  9  Caucasian  10  Caucasian  Student  11  Caucasian  Student  12  Caucasian  Student  13  Caucasian  Student  14  Caucasian  Unemployed  Run, Bike, Swim, Wts, Aerob.,skiing Business Women Run, Bike, Swim  Run, Walk, LaCrosse, Canoeing Fitness Instructor Run, Bike, Swim Student  202  Run, Bike, Row, Aerob, Swim, Wts Run, Bike, Swim, Row, Walk Run, Bike, Walk, Aerob, Stairm aster Run, Bike, Swim  Run, Bike, Swim, Walk  15  14 6  9 10 9  12  7  5 10  Appendix 22  Table of the distance run per week (km/week) by the female subjects throughout the study*.  Subject Code  Distance run per week (km/week)  1  43  2  55  3  39  4  42  5  45  6  53  7  20  8  40  9  38  10  10  11  21  12  49  13  45  14  20  203  *  VO  cn  V  »  5  o\  y  o  o\  ^ <* ^ £  ON  h  o  <n  et  a ON  o  oo  a  <u  3  v©  in  o  o  o m o cn cs ^ 00 ^  *  ©  ON  00  ON  © ©  >> n  o  ON  © Q >  00  © © m cn cs «0 ^ £  ON  >n cs © O > Q  CN  in  Ov  OO  m  S  Q  o  00  cn  ^  o  cs  r--  oo  ©  ©  Tt  o  oo cn © cs cn ^  o ao  m  CN o\  cn  CO  VO  ©  2 r» £ oo  O ^ Q  ©  ^ *  in ©  r- m vo  2  © cs 2 H ®  »  00  >n ©  © cs  >n  0  © cn  0  cn g g © © cs >n t> © O m ^ ~ m m™ Q Tt  Hi  13  S  ©  ft  VO  ©  vo  ^1 ^  CU  © © O m © cs i n > x2 i n  00 cn  00  ©  oo ©  5  •o o  ©  >n 00  S § § S•n cs  in  © cn  vo  «n  © in © cs © 00VOVoIQoN00^  S3  Q  >n r>  ©  H  © cn  ©  ' >n ->sfON  ON  cn ©  ^  ON  m ^  © m © r- vo  ©  in  m oo ^ .a '53 §'S  tu  on  •a  F3  a  ON  ©  ~  © © cs © © oo oo vo i n vo vo oo  00  •n —  © m ©r> vo Tcn t TJ-  .s tu tu  S in r-  Q  oo ^ m  ON  © ©  •n  i=; oo  in m  © ©  m  o oo 00  >n  oo m  c a> a, tu  a  £ l rt T3 O tU  • 1-1 *H  s  U  c N c n T t m v o t - ~ o o o N 2  204  cs cn  Tt  Q § * Q  XT.  &  CN  *  •a  Tt  r-  in o CO  ro  «  oo  "  o  •c  00  o  •o  r-  ON  >->  CN CN  •n  I  CM Ov Os  m  Tt  00  3 5  CN •n  •a  oo  Ov CO  r-  O  o oo  Tt  2  °  <n  w o  CN CN Tt  Tt  OS  VO  O <  Q  o Os  co OS OS  co *—I  Tt  rOs  o  P c > tu  tU 00  oo  ov  ft Q  0 01  p  ft  Q  co  I  r-  Ov VO CN  Os  O CO  in  5  S_i oo  3 2  Tt  o E3  o O  Ov  Tt  00  CO  o  2 ^  OS r00  o  ju  a  %  ft Q  VO VO  T?  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