Soy Isoflavones Modulated Antioxidant Defense Systems and Decreased Lipid Peroxidation in Rats and Humans

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1 Soy Isoflavones Modulated Antioxidant Defense Systems and Decreased Lipid Peroxidation in Rats and Humans By Chung-Yen Chen Dissertation submitted to the Graduate Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY In Human Nutrition, Foods & Exercise Dr. Raga M. Bakhit, Chair Dr. William E Barbeau, Ph.D. Dr. Allan H. Goldfarb, Ph.D. Dr. Golde Holtzman, Ph.D. Dr. Janet Walberg Rankin, Ph.D. August, 2001 Blacksburg, Virginia Keywords: isoflavones, antioxidant defense systems, exercise, rats, humans Copyright 2001, Chung-Yen Chen

2 Soy Isoflavones Modulated Antioxidant Defense Systems and Decreased Lipid Peroxidation in Rats and Humans by Chung-Yen Chen Chair: Raga M. Bakhit Human Nutrition, Foods & Exercise ABSTRACT Isoflavones have antioxidant activities in vivo, however, their antioxidative potential against oxidative stress initiated by exercise was not explored. The first study investigated the effect of high-genistin isoflavone (HGI) supplementation on erythrocyte antioxidant enzymes and tissues thiobarbituric reactive substances (TBARS) in acutely exercised oneyear old rats. All tissue genistein concentrations increased after exercise. Ingestion of HGI seemingly enhanced running time to exhaustion, and maintained glutathione peroxidase (GPx) and catalase (CAT) activities decreased due to exercise. The second study investigated the dose effect of HGI supplementation. Genistein concentrations were significantly higher (P<0.05) in tissues of rats fed the 1045 PPM HGI diet than in rats fed 522 or 209 PPM HGI diets and increased the glutathione (GSH)/total glutathione (TGSH) ratio (P<0.03). Reductions of the in vivo MDA concentrations (P<0.05) were observed only in the plasma of rats fed 522 and 1045 PPM HGI diets compared to those fed 0 PPM (-1.08, -0.82, and 0.03 µm, respectively). Therefore, isoflavones at PPM HGI diet have antioxidative effects in rats. The last two studies investigated the effect of isoflavone supplementation on the modulation of erythrocyte antioxidant enzyme activities, glutathione homeostasis, and other oxidative biomolecules in healthy young men undergoing 80%VO 2 pk exercise. In Study 3 exercise at 80%VO 2 pk increased oxidative stress which was best demonstrated by increased ii

3 superoxide dismutase (SOD) activity (16.5%), GSH/TGSH ratio, in vivo MDA (12.6%), plasma uric acid (4.9%) and ferric reducing/antioxidant ability (FRAP) ( 7.8%). Therefore, 30 minutes 80% VO 2 pk exercise induced oxidative stress in moderately active college men. In study 4, four-week HGI supplementation produced plasma genistein and daidzein concentrations of 499 and 415 ng/ml, which were significantly increased to 633 and 539 ng/ml by exercise (P=0.04 and P=0.05). Isoflavones significantly decreased in vivo preexercise plasma MDA (P<0.05), increased pre-exercise blood TGSH (P=0.01) and preexercise erythrocyte SOD activity (P=0.0006), and maintained the decreased activities of GPx due to exercise at pre-exercise levels. Results demonstrated that isoflavones had antioxidant activity in vivo under normal physiological conditions in healthy young men. They also maintaining GPx activity which was decreased due to exercise, however, isoflavones may not overcome all oxidative stress initiated by intense exercise. iii

4 DEDICATION To my parents Ma-Fu Chen and Huey-O Tsao. iv

5 ACKNOWLEDGEMENTS It has been five years since I followed my major advisor Dr. Raga M. Bakhit to conduct researches. There is no word which can express numerous thanks of mine to her. Her guidance and tremendous help have been changing my whole life. I would like to thank Dr. Allan Goldfarb for his unselfishness in sharing his knowledge with me. Even though he did not know me at all when we met at the first time, he patiently answered my questions. Without Dr. Golde Holtzman s instructions, I would have not completed my dissertation. Thank you for giving me some of your time whenever I sought your assistance to resolve the most complicated portion of dissertation, statistical analyses. To Dr. William Barbeau, thank you for your support and for your confidence and trust in me. Your words let me feel that I will have a bright future. To Dr. Janet Rankin, thank you for your time to share knowledge with me. Without your guidance and help, my research would not cross over Exercise Science field. I also want to thank Dr. Michael Houston. Without his unlimited support, I would not have completed my degree smoothly. I also wish to acknowledge Dr. Kathy Reynolds, Dr. David Moore, Janet Rinehart, Carolyn Harris, and David Gemmel. Without their unselfish and endless help, conducting experiments would be unachievable missions. I also like to express my thanks to all of my friends for their support, Vanessa, Sandy, Daly, Janet, and Bob, and all staffs in HNFE department. My final thanks go to my lovely girlfriend. Without her beside me, I do not know how I can pass all of these. Good luck to her and me! v

6 TABLE OF CONTENTS CHAPTER INTRODUCTION STATEMENT OF THE PROBLEM HYPOTHESES...4 CHAPTER LITERATURE REVIEW ATHEROSCLEROSIS RISK FACTORS OF ATHEROSCLEROSIS LDL OXIDATION Low density lipoprotein composition Evidence that LDL peroxidation leads to atherosclerosis Lipid peroxidation in the circulating LDL In vitro LDL oxidation by oxidants EXERCISE AND ROS GENERATION Free radical formation under normal physiological conditions Free radical-induced oxidation Free radical generation during exercise Biomarkers of oxidative stress induced by exercise Introduction Lipid peroxidation as a biomarker LDL oxidation ANTIOXIDANT DEENSE SYSTEMS AND ACUTE EXERCISE Antioxidant enzymes Antioxidants Vitamin C Glutathione homeostasis Uric acid ANTIOXIDANTS SUPPLEMENTATION AND EXERCISE ISOFLAVONES...23 vi

7 Introduction Metabolism of isoflavones Bioavailability of isoflavones Consumption of isoflavones in humans Antioxidative effect of genistein Other activities of isoflavones REFERENCES...29 CHAPTER ABSTRACT INTRODUCTION MATERIALS AND METHODS Animals Experimental design Sacrifice and sample collections Biochemical measurements Statistical analysis RESULTS Body weight, feed intake, and running ability Genistein concentrations Products of lipid peroxidation Activities of antioxidant enzymes in erythrocytes DISCUSSION SUMMARY REFERENCES...71 CHAPTER ABSTRACT INTRODUCTION MATERIALS AND METHODS Animals Experimental design Sacrifice, sample collections, and blood processing Biochemical measurements Statistical analysis...96 vii

8 4.4. RESULTS Feed intake and body weight Genistein concentrations Plasma lipid profile Lipid peroxidation in the plasma, liver, and muscle Total Glutathione and oxidized glutathione in whole blood DISCUSSION SUMMARY REFERENCES CHPATER ABSTRACT INTRODUCTION MATERIALS AND METHODS Subjects Exercise protocol Sample Collection Biochemical measurements Statistical analyses RESULTS DISCUSSION CONCLUSIONS REFERENCES CHAPTER ABSTRACT INTRODUCTION MATERIALS AND METHODS Subjects Study design, dietary intervention, and study timeline Peak oxygen consumption and submaximal exercise Sample Collection Biochemical measurements (the same methods as in chapter 5) Statistical Analyses RESULTS Nutrient intake and plasma isoflavones viii

9 Exercise intensities of initial and final submaximal exercises Exercise effect on biomarkers of oxidative stress The effect of isoflavone supplementation DISCUSSION CONCLUSIONS REFERENCES CHAPTER CONCLUSIONS CHAPTER FUTURE STUDIES APPENDICES APPENDIX A Progressive Exercise Test (VO 2 pk test) on Cycle Ergometer (Method) APPENDIX B Eighty Percent VO 2 pk Exercise on Cycle Ergometer (Method) APPENDIX C Total Glutathione and Oxidized Glutathione Analyses in Blood (Method) APPENDIX D Ascorbic Acid and Dehydroascorbic Acid Analyses in Human Plasma APPENDIX E LDL Separation from Plasma (Method) APPENDIX F Erythrocyte Superoxide Dismutase Assay (Method) APPENDIX G Erythrocyte Catalase Assay (Method) APPENDIX H Erythrocyte Glutathione Peroxidase Assay (Method) APPENDIX I Plasma Genistein and Daidzein Assay (Method) APPENDIX J Malondialdehyde by TBARS-HPLC Assay (Method) APPENDIX K Ferric Reducing/Antioxidant Ability of Plasma (FRAP) (Method) APPENDIX L ix

10 Hematocrit (Method) APPENDIX M Protein Assay-Sigma 5656 (St. Louis, MO) (Method) APPENDIX N Total Hemoglobin-Sigma 525A (St. Louis, MO) (Method) APPENDIX O Medical Health History Questionnaire (Subject Questionnaire) APPENDIX P Volunter Information (Subject Questionnaire) APPENDIX Q Food Intake Form (Subject Handout, to be filled out and returned) APPENDIX R How to Ccomplete Food Intake Form (Subject Handout) APPENDIX S Common Weights and Measures (Subject Handout) APPENDIX T Activity Diary (Subject Handout, to be filled out and returned) APPENDIX U Foods to Avoid (Subject Handout) APPENDIX W Compliance Survey (Subject Questionnaire) APPENDIX V IRB CURRICULM VITA x

11 LIST OF TABLES TABLE 3.1. GENISTEIN CONCENTRATIONS IN PLASMA, LIVER, AND GASTROCNEMIUS MUSCLE IN RATS.79 TABLE 3.2. MEAN LIVER CONCENTRATION OF MALONDIALDEHYDE BY DIETARY SUPPLEMENT AND EXERCISE...81 TABLE 3.3. MALONDIALDEHYDE (MDA) CONCENTRATIONS IN THE GASTROCNEMIUS MUSCLE.83 TABLE 3.4. THE ACTIVITIES OF SUPEROXIDE DISMUTASE (SOD) IN RBC IN RATS...85 TABLE 3.5. THE ACTIVITY OF CATALASE IN RBC 87 TABLE 3.6. THE ACTIVITY OF GLUTATHIONE PEROXIDASE (GPX) IN RBC...89 TABLE 4.1. THE COMPOSITION OF THE 0 PPM DIET TABLE 4.2. THE COMPOSITION OF ISOFLAVONE EXTRACT USED IN HGI DIET. 106 TABLE 4.3. GENISTEIN IN TISSUES AFTER FIVE WEEKS OF HGI SUPPLEMENTATION TABLE 4.4. LIPID PROFILES IN PLA SMA AFTER FIVE WEEKS OF HGI SUPPLEMENTATION TABLE 4.5. PLASMA MDA MEASURED BY HPLC AFTER FIVE WEEKS OF HGI SUPPLEMENTATION. 111 TABLE 4.6. MDA CONCENTRATIONS IN THE LIVER, AND GASTROCNEMIUS MUSCLE IN RATS FED HGI DIET. 113 TABLE 4.7. GLUTATHIONE, TOTAL GLUTATHIONE, AND OXIDIZED GLUTATHIONE CONCENTRATIONS IN WHOLE BLOOD AFTER 5-WEEK HGI SUPPLEMENTATION TABLE 5.1. SUBJECT CHARACTERISTICS AND DIETARY INTAKE 153 TABLE 5.2. ACTIVITIES OF ANTIOXIDANT ENZYMES IN RED BLOOD CELLS 154 TABLE 5.3. GLUTATHIONE CONCENTRATIONS AND GSH/TGSH RATIO IN WHOLE BLOOD156 TABLE 5.4. ASCORBIC ACID AND URIC ACID CONCENTRATIONS, AND FRAP VALUE IN PLASMA* 159 TABLE 5.5. MALONDIALDEHYDE CONCENTRATIONS IN PLASMA AND LDL*. 162 TABLE 6.1. CHARACTERISTICS OF SUBJECTS.202 TABLE 6.2. DIETARY INTAKE BEFORE TWO BOUTS OF SUBMAXIMAL EXERCISE.203 TABLE 6.3. IN VIVO PLASMA MDA IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION xi

12 TABLE 6.4. IN VIVO LDL-MDA IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE 6.5. EX VIVO LDL-MDA IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE 6.6. BLOOD TGSH IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE 6.7. BLOOD GSH IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE 6.8. BLOOD GSSG IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE 6.9. BLOOD GSH/TGSH RATIO IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION. 219 TABLE PLASMA ASCORBIC ACID IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE PLASMA TOTAL ASCORBIC ACID IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE PLASMA AA/TAA RATIO IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION. 225 TABLE PLASMA URIC ACID IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE PLASMA FRAP IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE ERYTHROCYTE SOD ACTIVITY IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE ERYTHROCYTE GPX ACTIVITY IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION TABLE ERYTHROCYTE CAT ACTIVITY IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION xii

13 TABLE OF FIGURES FIGURE 2.1. STEPS IN FORMATION OF ATHEROSCLEROTIC PLA QUE IN VIVO. SMC, SMOOTH MUSCLE CELL....6 FIGURE 2.2. SKELETONS OF CLASSICAL ISOFLAVONES, DAIDZEIN AND GENISTEIN...25 FIGURE 3.1. GENISTEIN CONCENTRATIONS IN PLASMA, LIVER, AND GASTROCNEMIUS MUSCLE IN RATS. MEANS FOLLOWED BY THE SAME SUPERSCRIPT WERE NOT SIGNIFICANTLY DIFFERENT AT THE 0.05 LEVEL OF SIGNIFICANCE EXPERIMENTWISE, USING A STUDENT S T- TEST...80 FIGURE 3.2. MEAN LIVER CONCENTRATION OF MALONDIALDEHYDE BY DIETARY SUPPLEMENT AND EXERCISE. MEANS FOLLOWED BY THE SAME SUPERSCRIPT WERE NOT SIGNIFICANTLY DIFFERENT AT THE 0.05 LEVEL OF SIGNIFICANCE EXPERIMENTWISE (TUKEY S HSD, SOKAL AND ROHLF, 1995). EXERCISE EFFECT WAS SIGNIFICANT, P= FIGURE 3.3. MALONDIALDEHYDE (MDA) CONCENTRATIONS IN THE GASTROCNEMIUS MUSCLE. THERE WAS A STATISTICAL DIFFERENCE DUE TO DIETS (P = 0.044) FIGURE 3.4. THE ACTIVITIES OF SUPEROXIDE DISMUTASE (SOD) IN RBC IN RATS. MEANS FOLLOWED BY THE SAME SUPERSCRIPT WERE NOT SIGNIFICANTLY DIFFERENT AT THE 0.05 LEVEL OF SIGNIFICANCE EXPERIMENTWISE (TUKEY S HSD, SOKAL AND ROHLF, 1995)..86 FIGURE 3.5. THE ACTIVITY OF CATALASE IN RBC. MEANS FOLLOWED BY THE SAME SUPERSCRIPT WERE NOT SIGNIFICANTLY DIFFERENT AT THE 0.05 LEVEL OF SIGNIFICANCE EXPERIMENTWISE (TUKEY S HSD, SOKAL AND ROHLF, 1995)...88 FIGURE 3.6. THE ACTIVITY OF GLUTATHIONE PEROXIDASE (GPX) IN RBC. MEANS FOLLOWED BY THE SAME SUPERSCRIPT WERE NOT SIGNIFICANTLY DIFFERENT AT THE 0.05 LEVEL OF SIGNIFICANCE EXPERIMENTWISE (TUKEY S HSD, SOKAL AND ROHLF, 1995) FIGURE 4.1. GENISTEIN IN TISSUES AFTER FIVE WEEKS OF HGI SUPPLEMENTATION. MEANS IN THE SAME TISSUE WITH THE DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT (P<.05), USING TUKEY S HSD (SOKAL AND ROHLF, 1995) xiii

14 FIGURE 4.2. LIPID PROFILES IN PLA SMA AFTER FIVE WEEKS OF HGI SUPPLEMENTATION. MEANS IN THE SAME MEA SUREMENT WITH THE DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT (P<.05), USING TUKEY S HSD (SOKAL AND ROHLF, 1995) FIGURE 4.3. THE DIFFERENCE OF PLA SMA MDA BETWEEN WEEK 0 AND 5. MEANS WITH THE DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT (P<.05), USING TUKEY S HSD (SOKAL AND ROHLF, 1995) FIGURE 4.4. MDA CONCENTRATIONS IN THE LIVER, AND GASTROCNEMIUS MUSCLE IN RATS FED HGI DIET. MEANS IN THE SAME TISSUE WITH THE DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT (P<.05), USING TUKEY S HSD (SOKAL AND ROHLF, 1995).114 FIGURE 4.5. GLUTATHIONE, TOTAL GLUTATHIONE, AND OXIDIZED GLUTATHIONE CONCENTRATIONS IN WHOLE BLOOD AFTER 5-WEEK HGI SUPPLEMENTATION. MEANS IN THE SAME MEASUREMENT WITH THE DIFFERENT LETTER WERE SIGNIFICANTLY DIFFERENT (P<.05), USING TUKEY S HSD (SOKAL AND ROHLF, 1995) FIGURE 4.6. THE RATIOS OF GSH/TGSH AND GSH/GSSG. MEANS IN THE SAME MEASUREMENT WITH THE DIFFERENT LETTER WERE SIGNIFICANTLY DIFFERENT (P<.05), USING TUKEY S HSD (SOKAL AND ROHLF, 1995) FIGURE 5.1. ACTIVITIES OF ANTIOXIDANT ENZYMES IN RED BLOOD CELLS. SOD, U/G HB; GPX, MMOL/S/MG HB; CAT, U/MG HB. MEANS IN THE SAME MEASUREMENT WITH THE DIFFERENT LETTER WERE SIGNIFICANTLY DIFFERENT (P<.05), USING PAIRED T-TEST.155 FIGURE 5.2. GLUTATHIONE HOMEOSTASIS IN WHOLE BLOOD. MEANS IN THE SAME MEASUREMENT WITH THE DIFFERENT LETTER WERE SIGNIFICANTLY DIFFERENT (P<.05), USING PAIRED T-TEST FIGURE 5.3. GLUTATHIONE RATIOS. MEANS IN THE SAME MEA SUREMENT WITH THE DIFFERENT LETTER WERE SIGNIFICANTLY DIFFERENT (P<.05), USING PAIRED T-TEST FIGURE 5.4. ASCORBIC ACID AND TOTAL ASCORBIC ACID CONCENTRATIONS IN PLASMA AND AA/TAA RATIO. A PAIRED T-TEST WAS APPLIED TO TEST THE SIGNIFICANCE OF CHANGES OF ALL MEASURED PARAMETERS DUE TO EXERCISE FIGURE 5.5. URIC ACID CONCENTRATION IN PLASMA AND FRAP VALUE. URIC ACID, MICROGRAM/ML; FRAP, µm. MEANS IN THE SAME MEASUREMENT WITH THE DIFFERENT LETTER WERE SIGNIFICANTLY DIFFERENT (P<.05), USING PAIRED T-TEST xiv

15 FIGURE 5.6. IN VIVO MALONDIALDEHYDE CONCENTRATIONS IN PLASMA AND LDL. MEANS IN THE SAME MEASUREMENT WITH THE DIFFERENT LETTER WERE SIGNIFICANTLY DIFFERENT (P<.05), USING PAIRED T-TEST FIGURE 5.7. IN VITRO LDL-MDA. MEANS WITH THE DIFFERENT LETTER WERE SIGNIFICANTLY DIFFERENT (P<.05), USING PAIRED T-TEST FIGURE 6.1. TIME OF THE STUDY FIGURE 6.2. PLASMA MDA. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< FIGURE 6.3. THE DIFFERENCE OF PLA SMA MDA BETWEEN PRE- AND POST-EXERCISE. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< FIGURE 6.4. LDL MALONDIALDEHYDE IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION 208 FIGURE 6.5. IN VITRO LDL-MDA IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION FIGURE 6.6. TOTAL GLUTATHIONE CONCENTRATION IN WHOLE BLOOD. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< FIGURE 6.7. THE DIFFERENCE OF TOTAL GLUTATHIONE BETWEEN PRE- AND POST-EXERCISE. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, USING TUKEY S HSD PROCEDURE (SALL ET AL., 2001), P< FIGURE 6.8. GLUTATHIONE CONCENTRATION IN WHOLE BLOOD. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< FIGURE 6.9. THE DIFFERENCE OF GLUTATHIONE IN WHOLE BLOOD BETWEEN PRE- AND POST- EXERCISE. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, USING TUKEY S HSD PROCEDURE (SALL ET AL., 2001), P< FIGURE OXIDIZED GLUTATHIONE IN WHOLE BLOOD. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< FIGURE GSH/TGSH RATIO IN WHOLE BLOOD. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< FIGURE PLASMA ASCORBIC ACID IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION. MEANS IN THE SAME DIET WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< xv

16 FIGURE PLASMA TOTAL ASCORBIC ACID IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION. MEANS IN THE SAME DIET WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< FIGURE PLASMA AA/TAA RATIO IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION FIGURE URIC ACID CONCENTRATION IN PLASMA FIGURE PLASMA FRAP IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< FIGURE THE DIFFERENCE OF PLA SMA FRAP BETWEEN PRE- AND POST-EXERCISE. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, USING TUKEY S HSD PROCEDURE (SALL ET AL., 2001), P< FIGURE SUPEROXIDE DISMUTASE ACTIVITY IN ERYTHROCYTES. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, P< FIGURE THE DIFFERENCE OF SOD ACTIVITY IN ERYTHROCYTES BETWEEN PRE- AND POST-EXERCISE FIGURE GLUTATHIONE PEROXIDASE ACTIVITY IN ERYTHROCYTES IN SUBJECTS BEFORE AND AFTER SUPPLEMENTATION FIGURE THE DIFFERENCE OF GLUATHIONE PEROXIDASE ACTIVITY BETWEEN PRE- AND POST-EXERCISE. MEANS WITH DIFFERENT LETTERS WERE SIGNIFICANTLY DIFFERENT, USING TUKEY S HSD PROCEDURE (SALL ET AL., 2001), P< FIGURE CATALASE ACTIVITY IN ERYTHROCYTES IN SUBJ ECTS BEFORE AND AFTER SUPPLEMENTATION xvi

17 ABBREVIATIONS USED IN THE DISSERTATION AA, ascorbic acid; AIN, American institute of nutrition; CAT, catalase; DHAA, dehydroascorbic acid; diff, difference as post-ex minus pre-ex; DMSO, dimethylsulfoxide; ELISA, enzyme-linked immunosorbent assay; FRAP, ferric reducing/antioxidant power; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, oxidized glutathione; HGI, high-genistin isoflavone extract; H 2 O 2, hydrogen peroxide; HPLC, high performance liquid chromatography; LDL, low-density lipoprotein; MDA, malondialdehyde; MPO, myeloperoxidase; O.- 2, superoxide; post-ex, post-exercise; pre-ex, pre-exercise; RBC, red blood cells; ROS, reactive oxygen species; SE, standard error; SOD, superoxide dismutase; SMC, smooth muscle cell; TAA, total ascorbic acid; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TGSH, total glutathione; UA, uric acid; and VO 2 pk, peak oxygen consumption. xvii

18 PRELUDE This dissertation is written in manuscript format where chapters 2-6 are complete papers each in its own right. Some redundancies are therefore inevitable, especially so in the introduction sections of closely topic related chapters. xviii

Y. Hong 1, S. Hong 1, Y. H. Chang 1, S. H. Cho 2. Republic of Korea,

INFLUENCE OF AN ORALLY EFFECTIVE SUPEROXIDE DISMUTASE (GLISODIN ) ON STRENUOUS EXERCISE-INDUCED CHANGES OF BLOOD ANTIOXIDANT ENZYMES AND PLASMA LACTATE Y. Hong 1, S. Hong 1, Y. H. Chang 1, S. H. Cho 2