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RELATIONSHIPS AMONG DIET, EXERCISE AND HEMATOLOGICAL PARAMETERS OF YOUNG COMPETITIVE RUNNERS VERSUS NONRUNNERS By Elaina Ryder A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTERS OF SCIENCE Department of Food Science and Human Nutrition 1986 ABSTRACT RELATIONSHIPS AMONG DIET, EXERCISE AND HEMATOLOGICAL PARAMETERS OF YOUNG COMPETITIVE RUNNERS VERSUS NONRUNNERS By Elaina Ryder HEmatological status and nutrient intake were evaluated for elite runners versus nonrunners, 8-16 years of age, in relation to developmental stage and physical capacity. Subjects were participants in a study on fitness and performance conducted by the Youth Sports Institute. Fasted venous blood samples were drawn for determination of hematological values, by automated procedures. Three day dietary records were evaluated for nutrient content using the Michigan State Nutrient Data Bank. Respiratory variables were obtained by treadmill testing. Runners were not at risk of iron deficiency anemia, as indicated by hematological parameters. Runners consumed greater energy, protein and iron intakes than controls. Hematological parameters were similar between iron-supplemented and non-supplemented subjects. No significant relationships were observed of physical capacity with hematological values. Percent of iron absorbed from a meal, calculated according to its bioavailability, was similar to subjects' tRDA for iron, if subjects were assumed to have 250 mg. iron storage levels. To my parent, Bernard and Janet Ryder, for all of their love and support throughout my graduate career. 11 ACKNOWLEDGEMENTS I Wish to express my sincere appreciation to Dr. Rachael Schemmel, my major professor, for all of her support of this research and her advice, criticism, and enthusiam throughout my graduate program and with this thesis. Sincere gratitude is extended to the members of my guidance committe: Dr. Vern Seefelt, for his tremendous counsel and support in data collection and analyses of this study; Dr. Thayne Dutson, for his words of encouragement and constructive suggestions throughout my master's program; and Dr. Al Sparrow, for his suggestions and advise in thesis development. Support of this project by an All University Research Initiation Grant, was appreciated. I want to also express appreciation to the many people who have had a part in completion of this study and my mater's degree: Dr. Van Huss and David Anderson, for their assistance in collecting the respiratory and lactate data. The professors, graduate students, and children who participated in the Youth Sports Institute, for their welcoming assistance and cooperation, and for providing research facilities. Dr. P. Ku, for his assistance and allowing me to use laboratory facilities for iron analyses. iii Jane Moeggenberg and Carol Conn for their tremendous assistance, cooperation, and enthusiasm in analyzing the data of this study. Charles Santerrne, for his assistance in computer programming and his friendship. Christi Steinbach, Bunee, Nika and Avy Kayne, Clare Hasler, Angi Frazer, Ann Carter, Louise Campbell, and and the girls in lab 311, for all of their support, encouragement, and their friendship. And a special thanks to my best friend, Susan DeVries, for all of her support, kindness, and time helping me through my studies. Finally, an enormous thankyou to my boyfriend, Steven Jurecki, for all of his patience, love, and words of encouragement throughout my graduate program. iv TABLE OF CONTENTS Page LIST OF TABLES O O O O 0 O O O O O O O O O O 0 v1. 1. LIST OF FIGURES . . . . . . . . . . . . . . . . x INTRODUCTION . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE General Metabolism of Iron . . . . . . . . . . . . 3 Iron Deficiency . . . . . . . . . . . . . . . 8 Iron Status Among Runners . . . . . . . . . . . . 17 Iron Nutriture . . . . . . . . . . . . . . . . 31 Iron Bioavailability . . . . . . . . . . . . . . 36 RESEARCH METHODS Subjects . . . . . . . . . . . . . . . . . . 48 Blood Analyses . . . . . . . . . . . . . . . . 49 Anthropometric Measurements . . . . . . 50 Performance Capability and Anaerobic Metabolism . . . . . 55 Nutrient Analysis . . . . . . . . . . . . . . . 56 Statistical Analyses . . . . . . . . . . . . 58 RESULTS Classification of Subjects . . . . . . . . . . . 61 HEmatological Profile . . . . . . . . . . . 63 Measurements of Physical Capacity . . . . . . . . . 66 Nutrient Intake . . . . . . . . . . . . . . . 68 Supplemental Iron Use . . . . . . . . . . . . . 71 Case Study Reports . . . . . . . . . . . . . . 72 Iron Bioavailability . . . . . . . . . . . . . . 74 Tables . . . . . . . . . . . . . . . . . . 77 Figure . . . . . . . . . . . . . . . . 105 DISCUSSION Classification of Subjects . . . . . . . . . . . 126 Hematological Profile . . . . . . . . . . . 128 Aerobic and Anaerobic Capacity . . . . . . . . . . 133 Dietary Evaluation . . . . . . . . . . . . . 140 Iron Supplementation . . . . . . . . . . . . . . 144 Case Study Reports . . . . . . . . . . . . . . 147 Iron Bioavailability . . . . . . . . . . . . . . 151 Page CONCLUSIONS 0 O O O O O O O O O O O O O O O O 158 LIMITATIONS OF THE STUDY AND RECOMMENDATIONS OF FUTURE RESEARCH 160 APPENDICES A. NHANES II Hematological and Iron Nutriture Median and 95% Range Values For Adolescents, 9-17 Years of Age . . 162 B. Monsen's Model; A Mbthod to Calculate The Percent of Absorbable Iron in a Meal . . . . . . . . . . . 163 REFERENCES 0 O I I O O O O O O O O O O O O 0 O 166 vi LIST OF TABLES Table Page 1. Stages of Iron Deficiency . . . . . . . . . . . 13 2. Ages, Heights, and Heights of Subjects in Phase II . . . 77 3. Ages, Heights, and Heights of Subjects in Phase III. . . 77 4. Gain in Height of Subjects from Phase II to III Classified According to Stage of Growth . . . . . . 78 5. Ages, Heights, and Heights of Subjects in Each Growth Category in Phase III . . . . . . . . . . . . 79 6. Relative Heights and Heights to Age of Subjects in Phase I I I O O O O O O O 0 O O O O O O O O 80 7. Relative Weights and Heights to Age of Each Growth Category in Phase III . . . . . . . . . . . . 8O 8. Hematological Value of Subjects in Phases II and III . . 81 9. Hematological Values of Each Growth Category in Phase III 82 IO. Correlation Between Gain in Height with Change in Hemoglobin and Hematocrit from Phase II to Phase III . . 82 11. Aerobic Capacity Measurements of Subjects in Phase II and Phase III 0 O O O O O O O O O O O O O O 83 12. Aerobic Capacity Measurements of Subjects in Each Growth Category in Phase III . . . . . . . . . . 84 Max with Hb., . and Phase III . . . . 85 13. Correlations Between Values of V0 Hct., and Plasma Iron in Phase 112 14. Anaerobic Capacity Measurements of Subjects in Phase III 86 15. Anaerobic Capacity Measurements of Each Growth Category 1“ Phase III 0 O O O O O O O O O O O O O 0 87 vii Table 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. Page Correlations of (1) VO2 Max Value with Maximum Lactate Levels 8 Total Lactate Produced; (2) Maximum Lactate Level with Hb., Hct., & Plasma Iron Values; & (3) Total Lactate Production with Hb., Hct., & Plasma Iron Values in Phase III . . . . . . . . . . . 88 Percentage of Energy Consumed from Protein, Carbohydrate, and Fat for Subjects in Phases II and III . . . . . . 89 Percent of Kilocalories Consumed from Protein, Carbohydrate, and Fat of Each Growth Category in Phase II and Phase I I I O I O O O O O O O O O O O O O 90 Iron Intake and VO2 Max Values for Users and Non-Users of Iron Supplement for MR and MC (Phase II) . . . . . 91 Iron intake and VO2 Max Values for Users and Non-Users of Iron Supplement for FR and FC (Phase II) . . . . . 92 Iron Intake and VO2 Max Values for Users and Non-Users of Iron Supplement for Males and Females (Phase II) . . 93 Iron Intake and VO2 Max Values for Users and Non-Users of Iron Supplement for MR and MC (Phase III) . . . . . 94 Iron intake and VO2 Max Values for Users and Non-Users of Iron Supplement for FR and FC (Phase III) . . . . . 95 Iron Intake and VO2 Max Values for Users and Non-Users of Iron Supplement for Males and Females (Phase III) . . 96 Changes in Hematological Measurements and V0 Max in Subjets while Taking Supplemental Iron as Co pared to when They Di d Not I O O O 0 O O O O O O O O O 97 Case Study Report on Subject with Low Hematological Measurements in Phase II . . . . . . . . . . . 98 Case Study Report on Subject with Low Hematological Measurements in Phase III . . . . . . . . . . . 99 Case Study Report on Subject with Low Hematological Measurements in Phase III . . . . . . . . . . . 100 Amount of Dietary Iron Absorbed as Calculated by Monsen's Model for Different Iron Storage Levels, 0 mg., 250 mg., and 500 mg. 0 O O O O O O 0 O O O O O O O O 101 Calculated Absorption of Iron Using Monsen's Model at 3 Levels of Iron Storage and 10% of the Actual Intake . . 101 viii Table Page 31. Percent of Subjects Absorbing Less than 1.2 mg. iron per day 0 O O O O O O O O O O O O O O 0 0 102 32. Correlation of Hemoglobin Values with 1RDA for Dietary Iron and z of 1.8 mg. Iron Absorbed, Calculated by Monsen's MdET O O O O O O O O O O O O O O O O O O 102 33. Dietary Intake of Nutrients Inhibiting Iron Absorption . 103 81. Factors for Estimating Percent Absorption of Dietary Iron at Each Iron Storage Level of 0 mg., 250 mg., and 500 mg. 165 ix LIST OF FIGURES Figures 1. Growth curve lines for the 3 growth categories; pre, peak, and post, plotted on a NCHS growth chart . . . . 2. Hemoglobin concentrations for individual subjects in each group of MR, MC, FR, and FC, for Phases II and 111. Median values of each group compared to the NHANES II median values; 14 g/dl for males and 13.4 g/dl for fanales’ 12 - 14 years 01d 9 o o o o o o o o o o 3. Hematocrit concentrations for individual subjects in each group of MR, MC, FR, and FC, for Phase II and III. Median values of each group compared to the NHANES II median values; 40.5% for males and 39% for females, 12 " 14 years 01d 0 o o o o o o o o o o o o o 4. Plasma iron concentrations for individual subjects in each group of MR, MC, FR, and FC, for Phase III. Median values of each group compared to the NHANES II median values; 95 mcg/dl for males and 96 mcg/dl for females, 12 - 14 years 01d 0 O O O O O O O O O O O O I O O 5. Mean daily energy consumption, for MR, MC, FR, and FC for Phases II and III 0 O O O O O O O O O O O O O 6. Mean daily energy consumption, for subjects categorized into pre- peak- and post growth spurt for MR, MC, FR, and FC for Phase III . . . . . . . . . . . . . . 7. Mean kcal. intake /kg. body weight for MR, MC, FR, and FC for Phases II and III I O O I O O O O O O O O 8. Mean kcal. intake/kg. body weight for subjects categorized into pre-, peak-, and post- growth spurts for MR, MC, FR, and FC for Phase I I I O O I O O O I O O O O O O 9. Mean daily protein intake, for MR, MC, FR, and FC for Phases II and I II C O O O O I O O O C O O O O Page 53 105 107 109 111 111 113 113 115 Figures 10. 11. 12. 13. 14. 15. 16. 17. 18. Mean daily protein intake, for subjects categorized into pre-, peak-, and post- growth spurts for MR, MC, FR, and PC for Phase III . . . . . . . . . . . . . Mean daily dietary iron intake, for MR, MC, FR, and PC for Phases II and III . . . . . . . . . . . Mean daily dietary iron intake, for subjects categorized into pre-, peak-, and post- growth spurt for MR, MC, FR, and FC for Phase III . . . . . . . . . . . . Subjects were divided into 8 categories based upon their mean daily iron intakes in mg. This figure illustrates the percentage of subjects who fell within each category in Phase II . . . . . . . . . . . . . . . Subjects were divided into 8 categories based upon their mean daily iron intakes in mg. This figure illustrates the percentage of subjects who fell within each category in Phase III . . . . . . . . . . . . . . Mean daily total protein, animal protein, and plant protein intakes for MR, MC, FR, and FC, for Phase III . Percent of the mean daily dietary iron intake associated with dietary heme, for MR, MC, FR, and FC, for Phase III Mean dietary Vitamin C intake, for MR, MC, FR, and FC, for Phase III . . . . . . . . . . . . . . Comparison of the percent of total dietary iron intake calculated to be absorbed at the specified body iron storage levels (0 mg., 250 mg., and 500 mg.), according to Monsen's Model (18), for each group with the 10% absorption assumed for determining the RDA for iron . xi Page 115 117 117 119 119 121 123 123 125 INTRODUCTION Iron deficiency anemia is the most common nutritional problem among adolescents (1). There is an increased requirement for iron for this age group because of rapid growth and an increase in hemoglobin production for expanding blood volume, occuring during sexual maturation (2). Combined with this increased need, a low intake of dietary iron by adolescents has been frequently reported in nutritional surveys (3-6). Several investigators (7-11) have reported that mature, elite runners have a greater tendency to develop anemia than less active individuals. Possible causes for this, as suggested in these investigations, include; sports anemia, disturbance in iron absorption, greater iron loss through excess sweating, and hemoglobinuria and hematuria. Thus far, there have been no studies conducted comparing the hematological status of adolescent runners and non-runners. The question of iron supplementation of the diet has aroused considerable controversy during the recent years. Conflicting results have been reported in studies conducted to determine the effectiveness of prophylactic iron supplementation in athletic and sedentary individuals (12-15). More research must be done to determine if iron supplementation may or may not be necessary for athletes. Iron absorption from foods depends not only upon the amount of 1 iron supplied, but by the nature of that iron and the composition of the meal in which it is consumed (16). Tbtal iron intake, therefore, provides only a rough approximation of the amount available for absorption (17). Monsen (18) developed a model to calculate the amount of absorbable iron in the diet based on the iron status of the individual and the amount of enhancing factors, meat and ascorbic acid, present in the meal. The main objective of this method was to classify the quality of iron in a meal as having high, medium, or low availability. The purpose of this study was to determine if a sub-population group of elite runners, 8-16 years of age, had a higher incidence of marginal iron status than normal teenage controls of similar age and gender. The objectives of this study were: -- To assess the hematological status and nutrient intake in relation to developmental stage and physical capacity over a two year period. -- To compare the hematological status with quantitative changes in growth, physical capacity, and anaerobic metabolism. -- To determine the effect of prophylactic iron supplementation on hematological status and performance capability. -- To evaluate the quality of subjects' diets in terms of the bioavailability of iron. REVIEW OF LITERATURE GENERAL METABOLISM OF IRON IRON UTILIZATION AND STORAGE The nutritional requirement for iron in humans is derived from the central role that this metal plays in the energy metabolism of cells. Most of the body iron exists in complex forms bound to protein, either as porphyrin or heme compounds including; hemoglobin, cytochromes a, b, and c and P-450, and myoglobin. Iron also exists as nonheme, protein-bound compounds including; ferritin, transferrin, hemosiderin, and flavoprotein enzymes (19). The total body iron in an adult (70 kg. male) is approximately 4 to 5 grams of which 65% is bound as hemoglobin, 15-20% as ferritin, 3-5% as myoglobin, and the remainder as hemosiderin and iron-containing enzymes (20). Iron occurs in blood bound to hemoglobin in the erythrocytes and to transferrin in the plasma in a ratio of nearly 1000:1 (19). Hemoglobin consist of four ferroprotoporphyrin or "heme" moieties linked to four polypeptide chains. Each heme can reversibly bind to one molecule of oxygen functioning as a carrier to supply oxygen to all cells in the body. Myoglobin contains one heme moity and has a higher affinity for oxygen than hemoglobin. This heme compound is found in muscle cells. It functions as an oxygen store, releasing oxygen to cytochrome oxidase when the supply of oxygen is insufficient for the needs of the tissues (21). Mitochondria contain an electron transport system which transfers electrons from substrates to molecular oxygen - with the simultaneous generation of adenosine triphosphate (ATP). The cytochromes are components of this system. The iron atoms of cytochromes and iron-sulfer enzymes are alternately oxidized and reduced in the process of electron transport. The iron sulfur proteins or flavoprotein enzymes consist of non-heme iron in the active center, which also participate in the electron transport chain (21). Transferrin is a glycoprotein with two almost identical iron- binding sites - each capable of binding one atom of ferric iron. Transferrin serves as the principle carrier of iron in the blood, and therefore, plays a central role in iron metabolism. In normal individuals only 30-40% of the transferrin carries iron, the remainder being known as the latent iron-binding capacity (19). The reserve or storage iron of the body occurs predominantly as ferritin and hemosiderin. These occur widely in the tissues, with the highest concentrations normally present in the liver, spleen, and bone marrow. The two compounds are chemically dissimilar although intimately related in function. These compounds are involved in the maintenance of iron homeostasis. When the supply of dietary iron becomes inadequate, iron is mobilized from ferritin and hemosiderin and serves to maintain the production of hemoglobin and other iron compounds with known metabolic functions. Not until these products become restricted is there likely to be any impairment of body function (1). The main factor affecting the relative distribution of iron between ferritin and hemosiderin in mammals is the total storage iron concentration. When total storage iron in the liver and spleen is below 500 mg./g. of tissue, more iron is stored as ferritin than as hemosiderin. When the storage level is above 1000 ug./g., more is stored as hemosiderin (1). Ferritin is the soluble iron storage protein found in all cells of the body. Ferritin is a ferroxidase, +2 to Fe+3 during its incorporation into catalyzing the oxidation of Fe the iron core. Small quantities of ferritin are present in the erythrocytes, serum, and leukocytes. The levels of ferritin in serum vary with the iron status of the individual and with certain disease states. This form represents only 0.2-0.4% of the serum iron normally present in the adult (21). Hemosiderin is a term applied to iron which, after staining with potassium ferrocyanide, can be seen as blue granules in sections of liver or bone marrow. Histochemical examination of aspirated samples of bone marrow provides a useful index of body iron stores (19). IRON ABSORPTION The three main phases in the absorption of iron from the gut include: the intraluminal phase, where food is digested by the gastric and pancreatic enzymes and iron is released in a soluable form; the mucosal phase, in which iron is taken up by the mucosal cell and transported across to the serosal side or retained as ferritin; and the corporeal phase, in which iron is taken up by transferrin in plasma on the serosal side of the mucosal cell and carried to liver and hemapoietic tissues (22). The absorption of iron is affected by: age, iron status, and state of health of the individual; conditions within the gastrointestinal tract; the amount and chemical form of the iron ingested; and the amounts and proportions of various other components of the diet, both organic and inorganic (19). The most important known stimuli to iron absorption include the rate of erythropoiesis and the levels of tissue iron stores. These two factors regulate absorption particularly at the level of serosal transfer. The way these stimuli inform the duodenum to transfer appropriate amounts of iron into the plasma is unknown (20). It appears likely that homeostatic mechanisms within the body affect the populations of brush border receptors - according to iron status - at the time of mucosal cell formation. Changes in transport across the serosal surface, occurring with a somewhat shorter time lag, respond more quickly to changes in iron status. Iron can also enter the mucosal cells from the plasma and pass back into the lumen by active extrusion, particularly in the lower part of the small intestine (23). The maximal absorption of iron takes place in the duodenum. The efficiency decreases from the proximal to the distal part of the small intestine. The amount of iron transferred from the gut lumen to the mucosa depends upon the abundance of receptors on the brush border. The receptor population increases in iron deficiency, the increase being more in the distal than in the proximal part of the intestine (22). Once taken up by the mucosa, some of the iron passes rapidly into the circulating plasma (21). The excess iron in the mucosal cell, not transferred to plasma transferrin on the serosal side, is taken up by apoferritin and stored as ferritin (22). Cellular iron may enter the body to meet current body requirements or may remain within the cell to limit mucosal uptake of iron. This iron may be excreted when the cells are sloughed from the villus. In iron-deficient subjects little iron is incorporated into these cells from body stores so that absorption is enhanced and excretion is diminished (20). .159! METABOLIC PATHWAYS The major movement of iron in the body is unidirectional; absorbed iron is attached to transferrin, which delivers it to the erythroid precursors in the bone marrow. Iron is utilized to form hemoglobin, which in turn, is then incorporated into the red blood cell. The hemoglobin remains within the red cell for its 120-day life-span. The red blood cell is then phagocytized in the reticuloendothelial system and the iron is released. Approximately 85% of the iron derived from the catabolism of red blood cells is promptly returned to the plasma. The remaining iron is stored in the reticuloendothelial cell (20). The subsidiary metabolic pathways involve the plasma iron attaching to transferrin and then being delivered to cells throughout the body for the synthesis of ferritin, hemosiderin, myoglobin, and the iron-containing enzymes (19). IRQM’EXCRETION Previously it was believed that the quantity of iron in the body was controlled solely by regulation of absorption and that excretion played a passive role. However, most cells contain iron somewhat in proportion to the quantity of iron in body stores. Thus the daily obligatory loss of cells from skin and gut secretions - such as bile and sweat - provide a limited but selective loss of body iron (20). The major rate of loss of iron was found to be through the gastrointestinal tract with mean losses of 0.38 mg. per day from blood loss, 0.25 mg. per day from the bile, and 0.1 mg. per day from exfoliated epithelial cells. Losses in the urine were approximately 0.1 mg. per day (21). The total amount of iron lost daily in the sweat depends on the individual, the ambient temperature, and the dermal cell content in sweat loss. The average loss of iron through dermal cell loss of a healthy adult has been assessed as about 0.5 mg. per day. The total quantity of iron lost in the urine, feces, and sweat (excluding iron from the dermal cell in sweat) amounts to 0.6 - 1.0 mg. per day in most individuals. A loss of this magnitude is appreciable when it is realized that the average amount of iron absorbed from ordinary mixed diets is only 1.0 - 1.5 mg. per day (19). IRON DEFICIENCY PREVALENCE Iron deficiency anemia is the most common nutritional disease among adolescents. Several studies have indicated a high prevalency among teenage males - more so than females before the onset of menses. According to the 1968-1970 U.S. Public Health Service, Ten State Nutritional Survey (3), 5 - 10% of teenagers had below normal hemoglobin and hematocrit levels. In that survey, the normal ranges for hemoglobin were 14.0 i Zg/dl for females and 16.0 i 2 g/dl for males. The normal ranges for hematocrit were 42‘: 5% for females and 47.: 5% for males. In the 1970-1972 U.S. Department of Health, Education, and Welfare, Health and Nutritional Examination survey (NHANESI) (24), it was reported that 10% of the boys and 5% of the girls were iron deficient based on hemoglobin, hematocrit, serum iron, and serum transferrin levels. In the 1976-1980 NHANESII survey (25), 2-3% of the male and 4-6% of the female adolescents had hemoglobin and hemacrit values indicating iron deficiency anemia. In other studies (26), the incidence of iron deficiency was reported to be in the range of 10 - 27% for female and 13 - 50% for male adolescents. Iron deficiency anemia, as a term, has been almost interchangeable with nutritional anemia and has represented the most prevalent deficiency among children in the U.S. Its frequency has even prompted the suggestion that it has been the most frequent disorder seen in clinical medicine (27). DESCRIPTION 95 159! DEFICIENCY AND METHODS 95 DETECTION Iron deficiency anemia is a progressive condition of negative iron balance that is created when the physiological demand for iron is in excess of the iron ingested. Exogenous iron loss, iron absorbed from food, and iron stores available in the body are factors that affect iron balance. Iron deficiency can be due to one or more conditions such as acute or chronic blood loss or destruction, decreased iron intake, impaired absorption and/or increased requirements due to rapid growth (1). Iron deficiency is considered to be a state in which the iron supply is inadequate to permit normal synthesis of essential iron compounds (28). Although usually considered benign, iron deficiency anemia may have serious debilitating effects. These include: decreased resistance to infection, impaired immune response, symptoms of irritability and fatigue, alterations in temperature regulations, a diminished capacity for work and activity, and lowered intellectual motivation and performance (27,29,30). Functional gastrointestinal abnormalities are also associated with iron deficiency including; a reduction of acid secretion by the stomach, an impairment in iron, fat, and xylose absorption, an occult gastrointestinal bleeding in infants, and varying degrees of histologic changes in the duodenal mucosa (29,31). In the past, physicians and investigators have been too concerned with the 10 circulatory hemoglobin level and not enough with the other effects associated with tissue iron deficiency. More recent investigations have indicated abnormalities associated with muscle function and resistance to infection in iron-deficient animals and man. It seems likely that an element such as iron, which is involved in so many essential tissue reactions, would be found to be vital to body functions other than oxygen transport. Such tissue effects of iron deficiency require further examination (28). Iron depletion exists in varying degrees which extend from the mild depletion of iron stores to the development of a severe anemia (32). If the body goes into negative iron balance, iron is removed from the body stores (ferritin and hemosiderin) for metabolic needs. Simultaneously, iron absorption is enhanced and there may be some increase in the iron binding capacity of the plasma (33). During this phase, plasma iron concentration is not appreciably altered. When iron stores become exhausted, however, plasma iron levels fall and erythropoiesis is curtailed. Normocytic and normochromic anemia is often seen with iron deficiency when the anemia is mild or developing rapidly (32). No single iron parameter monitors the entire spectrum of iron status (33). These parameters of iron status reflect changes in different body iron compartments and are affected at different levels of iron depletion. It is convenient to define iron deficiency as progressing through three stages (34). Distinctions between the three levels of iron deficiency are entirely arbitrary, because iron stores in a population form a continuum, ranging from severely iron deficient to iron overload (33). The least severe stage of iron deficiency, "iron 11 depletion”, occurs when iron stores (in the liver, spleen, and bone marrow) fall to less than 100 mg. as indicated by a marked reduction in ferritin and hemosiderin (30,33,35). Quantitative estimates of iron status can be made from tissue by making chemical assessments of samples removed from bone marrow or by making determinations of the serum ferritin concentration of a blood sample (33,36). The serum ferritin concentration is directly proportional to the level of available storage iron in the liver and bone marrow. Levels of serum ferritin are inversely related to iron absorption; absorption increases when iron stores are depleted (34). Measurement of serum ferritin allow the estimation of iron stores by noninvasive radio or enzymatic immunoassay procedures (35). A low concentration of serum ferritin is characteristic only of iron deficiency. However, when inflammatory disease and iron deficiency coexist, serum ferritin values may be within the normal range (1). Once the iron stores have been depleted, serum ferritin levels will not reflect advanced stages of iron deficiency (35). With continued iron loss, iron stores become exhausted and the second stage of iron deficiency erythropoiesis ensues. During this stage, the level of the heme precursor (protoporphyrin) rises in the red cells. This rise indicates an insufficient supply of serum iron for hemoglobin synthesis and red blood cell production (34). Indicators of this stage include an increased level of free erythrocyte protoporphyrin (FEP) and total iron binding capacity (TIBC), and a decreased level of serum iron and percent transferrin saturation (33,36). The FEP can be measured rapidly by a simple fluorescence assay using a fluorometer. The serum iron and TIBC are 12 most commonly measured by atomic absorption or spectrophotometric techniques. The percent of transferrin saturation is derived simply by dividing the serum iron concentration by the TIBC value and multiplying by 100 (1). During the final stage of iron deficiency, "iron deficiency anemia”, the diminishing iron supply will further impair red cell production. This phase is identified by a significant fall in the circulating hemoglobin and alterations in the hematological indices (33,36). Laboratory diagnosis can be made by analyzing the results from a complete blood count, including a differential smear, and by measuring the concentrations of the hematological parameters. If the analysis of the blood smear shows small cells (microcytic) and undercolored cells (hypochromic), the diagnosis usually is iron deficiency anemia. The hematological parameters measured include hemoglobin, hematocrit (or packed cell volume - PCV), red blood cell count (RBC), and mean cell hemoglobin concentration (MCHC), mean cell volume (MCV), and mean cell hemoglobin (MCH) (33). The concentration of hemoglobin is measured by diluting a blood sample with a solution that converts the hemoglobin to cyanmethemoglobin, which is then quantified spectrophotometrically (37). The hematocrit is measured by centrifugation of a minute amount of blood that has been collected in a heparinized capillary tube. The hematocrit is then calculated by comparing the height of the column of packed red cells to that of the plasma (38). Electronic coulter counters are commonly used to accurately measure red blood cell count, MCV, MCH, and MCHC directly (1). Table 1 illustrates the development of iron deficiency and the various parameters indicating each level of iron status. 13 Table 1. STAGES OF IRON DEFICIENCY STAGE METHOD OF DETECTION PHYSIOLOGICAL CONSEQUENCES 1. Iron Depletion 2. Iron Deficiency Erythro- poiesis 3. Iron Deficiency Anemia Serum Ferritin Plasma Iron TIBC Transferrin Saturation Hemoglobin RBC Count Hematocrit MCV MCHC Depletion of iron stores in liver, spleen, and bone marrow. Iron stores have been depleted; levels of iron carried in the plasma decrease and transferrin formation in the liver increase. Total iron-binding capacity increases to levels of 400-500 ug/dl. Percent saturation of transferrin with iron falls from a mean of 30% to about 15% to 18%. Hemoglobin concentration falls below 12 g./dl. The degree of iron deficiency anemia can be evaluated with additional blood data. 14 In a recent survey of 1,564 subjects, living in Northwestern U.S. (33), it was observed that if only one of the three parameters - serum ferritin, transferrin saturation, or hemoglobin (each being from a different stage of iron deficiency) - was abnormal, the prevalence of anemia was only slightly higher (10.9%) than in the population as a whole (8.8%). However, when any two of these three parameters were abnormal, the prevalence of anemia increased to 28% and when all three parameters were abnormal, to 63%. Thus, the cause of anemia can be reasonably attributed to iron deficiency only when at least two iron parameters fall within the iron deficient range. The magnitude of analytic errors and the within subject biological variations are less than 4% for hemoglobin, hematocrit, and red cell indices (39). Higher coefficients of variation are characteristic of serum iron, TIBC, serum ferritin, and erythrocyte protoporphyrin (41). Remarkably consistent results with less than 2% experimental error) can by obtained by experienced laboratories for hemoglobin, hematocrit, and red blood indices (39). This facilitates the detection of mild anemia. Furthermore, the relatively small biological variations in these laboratory measurements make it easier to distinguish even a relatively small response to therapy from a random fluctuation. In of serum ferritin, TIBC and eythrocyte protoporphyrin, analytic variation can be drastically decreased by the use of automated equipment, which decreases environmental contamination. Variations due to biological factors are much greater than analytic variations with an automated method. The variations due to diurnal factors can be minimized by sampling in the morning or early afternoon; values can normally fall to very low levels at night. There is an impression that the 15 biological variation in measurements of iron diminishes in iron deficiency, resulting in less fluctuation of low values. With improvements in methodology, a major remaining problem will relate to the overlap of subjects, making it difficult to identify individuals with mild iron deficiency, but not a major obstacle when characterizing groups of subjects (39). Perhaps the most reliable criterion of iron deficiency anemia is the hemoglobin response to an adequate therapeutic dose of iron. A therapeutic trial allows the recognition of an individual whose hemoglobin value, although within the reference range, is low for him/her (1). When screening a large segnent of a population, hematocrit and hemoglobin are the conmon procedures to determine iron status (34). Therapy of the anemia itself involves reversal of the sequence of iron depletion, first repletion of functional body iron compounds and then of iron stores. Repletion of the stores will occur slowly when iron is given orally. This form of iron therapy must, therefore, be continued for many months after the hemoglobin level has returned to normal to fully replete the stores. In most serious cases of iron deficiency, the subject will usually recieve injections of an iron dextran to allow a more rapid replacement of iron stores (32). ADOLESCENCE The demands of the body for iron are greatest during three periods - the first two years of life, the period of rapid growth and hemoglobin increase of adolescence, and throughout the child-bearing period in women (19). The acceleration of growth rate and weight gain, particularly during the years of sexual maturation, impose increased requirements for iron, primarily for the production of 16 hemoglobin (1). Iron deficiency is a common finding among adolescents of both genders due to the increased need for iron and the low iron content of the foods most commonly eaten by this age group. This nutritional disease is more prevalent in boys than in girls because of their greater expansion of blood volume and lean body mass associated with growth (40). For example, during the peak year of their adolescent growth spurt, boys gain an average of ten kilograms. This can be calculated to require a net increase of approximately 300 mg. of iron merely to maintain a constant concentration of hemoglobin in an expanding blood volume (I). In a group of 14,000 subjects who had a constant iron intake of 6 - 9.5 mg. per day after infancy, the rate of anemia increased from 2% in the 11 year old male to 30% in the 15 year old male largely due to growth in lean body mass. In the girls, the rate of anemia was not as great, but approximately two times higher in the 15 year old than in the 11 year old females (40). In the adolescent girls, iron needs are also large, but their growth rate does not peak as sharply as in boys. The maximum yearly weight gain is somewhat less than in boys and the concentration of hemoglobin in girls increases only slightly during this period. The greatest average weight gain of 9 kg. per year in girls requires approximately 280 mg. iron for the maintenance of a constant concentration of hemoglobin (1). The onset of menses usually follows the peak of adolescent growth. The median menstrual blood loss of approximately 30 ml. per menstrual period in 15 year old girls involves a net loss of about 175 mg. of iron per year (1). Adolescent females are considered to be nutritionally vulnerable because of the rapid growth rate combined with marginal nutrient intake and menstrual 17 loss. The three major factors influencing the status of iron nutrition throughout the adolescent period for both males and females include; the amount and bioavailability of iron consumed, the rate of body growth, and the amount of iron loss. Each factor affects the amount of iron available for both metabolism and storage (34). Until relatively recently, young adults were the usual basis for reference ranges of laboratory tests. Increasingly, the use of age- specific criteria for children has become accepted, particularly in relation to hemoglobin, hematocrit, RBC indices, serum iron, TIBC, and transferrin saturation (41). One basis for this conclusion is related to the fact that the ranges of laboratory values in children, with the exception of erythrocyte protoporphyrin, tend to be narrower than those in adults (25). Laboratory results from the second National Health and Nutrition examination survey (NHANES II) have been used to define age-related changes in values used in the diagnosis of anemia and iron deficiency. Analyses included hemoglobin, hematocrit, RBC, red cell indices, serum iron, TIBC, transferrin saturation, and erythrocyte protoporphyrin which had been uniformly performed on a representative sample of 15,093 subjects, 1 to 74 years of age. The median value and the 95% range of each parameter measured for age categories, 9 to 11, 12 to 14, and 15 to 17 year old males and females are listed in Appendix A (25). IRON STATUS AMONG RUNNERS SPORTS ANEMIA The reduction of red blood cells during strenuous exercise has been recognized in human beings, dogs, and rats. The anemia occurs at an early period of physical training but eventually disappears after 18 one to three weeks from when the exercise was initiated. The blood properties return to the initial level shortly after the exercise is discontinued. In a normal individual, a practically constant balance is maintained between blood destruction and blood formation. This is not the case when subjects have been kept under sedentary conditions for a long time and then suddenly subjected to a strenous exercise for several days. In this situation, the blood cells are destroyed more rapidly than the hematopoietic tissue can replace them resulting in a marked fall in volume of red blood cells and circulating hemoglobin This exercise-induced reduction of red blood cell has been termed ”sports anemia" (42). Increased destruction of red blood cells during strenous muscular exercise has been known since the beginning of this century. Studies to determine the mechanism were first done on dogs by 0.0. Brown in 1922 (7). Brown postulated that the reduction in resistance of the red cell membranes was caused by the wear and tear of increased circulation through the capillaries. J.E. Davis concluded (43) that this decreased resistance in the membranes was due to the high body temperature brought on by heavy muscular work. Sports anemia is characterized by a transient decrease in hemoglobin concentration, RBC and PCV, but rarely results in clinical anemia. The red blood cells remain normocytic and normochromic (8). Associated with the red cell reduction is a concomitant decrease in serum protein (42). These reductions are not merely due to the hemodilution which results from an expanding plasma volume, which commonly occurs in athletes while training for a sport. There is an absolute reduction of hemoglobin as indicated by a significant 19 decrease in the total circulating hemoglobin detected after one week of training. An observed increase in osmotic fragility of the erythrocyte membrane has been associated with this reduction in hemoglobin and hematocrit This has been reported to occur as early as the first day of training, and remaining elevated throughout an entire seven-day, exercise period. The decrease in the integrity of the red blood cell membrane has been attributed to the increased circulation rate, temperature and acidity of the blood and the greater compression on the cell which results from training (44). Although the lysis of erythrocytes occurring with severe work has been generally widely accepted at the present time, it has not been possible to clearly explain the exact physiological reasons for this increase in intravascular hemolysis. A number of theories have been proposed as reasons for this temporary disorder (44). Shirahi demonstrated (45) that this increase in fragility and reduction of red cells during exercise could be prevented by removing the spleen in dogs. In this report, it was also shown that the effete red cells - with cell membranes having higher osmotic fragility resulting from the exercise - appearing in exercising dogs could be normalized by incubating these in the plasma of a resting or splenectomized dog. Shirahi concluded that the sports anemia was caused by the liberation of some hemolysing factor (termed lysolecithin) from the spleen. When a subject has undergone strenous muscular exercise, an increase in epinephrine secretion would be promoted by the stress. This would cause an acceleration in the contraction of the spleen allowing the hemolysing factor to flow out into the circulating blood. This has been the proposed mechanism which initiates sports anemia (45). 20 Several investigators believe that the increase in lysis of red cells may be an adaptive process to strenous muscular exercise. The heme component of the destroyed red cells and the serum protein may be utilized by the muscles to meet the increased demand for protein metabolism during exercise (42,44). Yoshimura (7) demonstrated that the myoglobin in limb skeletal muscle of rats exercising for two weeks increased as compared with no change in amount in resting controls. Thus, he concluded that the hemoglobin in red cells has been utilized to produce muscle protein and myoglobin as well as new red cells. Hiramatsu (7) found that after one week of hard physical training, the spherical index of the subjects' red blood cell had increased; thus decreasing the fragility of the cell. This may be regarded as an adaptive reaction to promote growth or hypertrophy of muscles and regeneration of new and strong red cells capable of withstanding strenous physical training. In another study (45), Hiramatsu found a faster decay rate in the specific activity of hemin 59Fe in red blood cells in exercising versus sedentary rats, indicating an increase in destruction of labeled red blood cells. In the training group, the rate of incorporation of hemin 59Fe was sharply accelerated in all tissues (especially in the skeletal and heart muscle, spleen, and bone marrow where hemoglobin or myoglobin are contained) as compared to that in the sedentary control. The rate of incorporation of 59Fe was much greater from red blood cell hemin than from serum injected into the muscles, spleen and bone marrow. These results demonstrated how 59Fe could be incorported more easily into these organs when obtained from hemoglobin than from serum. Therefore, the hemoglobin iron may facilitate the expansion of muscles and synthesis of new red blood 21 cells associated with training. Thus, Hiramatsu had presumed that the hemin molecule, labeled with 59Fe hemoglobin, was directly incorporated into the myoglobin in the muscle. There seems to be a temporary alteration of priorities for iron needs during exercise. Increased levels of exercise stimulate the increased production of myoglobin in the hypertropic muscle. A greater amount of iron would be required to synthesize the myoglobin, taking precedence over erythropoiesis, so oxygen delivery to exercising muscle would not be compromised. If iron was relatively unavailable, the new generation of red blood cells might be produced without a full complement of hemoglobin (46). A low-grade runner's hemolysis could create and sustain a negative iron balance, especially in subjects with low iron stores and low dietary iron absorption. In a recent study of 16 marathon runners (47), a poor overall correlation had been observed between hematocrit and performance, but the faster runners had significantly higher pre- race hematocrits. (mean 49%) than the slower group (mean 45%). These results indicate that runner's hemolysis may prevent the attainment of optimal red cell mass for maximal race performance. PREVALENCE 9E LOW IRON STATUS AMONG ATHLETES Runners have a greater tendency to develop anemia than less active individuals due to several factors associated with the exercise. In one investigation (8), the effect of running on indices of iron status in young female cross-country runners was studied during their training and competitive season. The runners experienced sports anemia as indicated by a decrease in hemoglobin and PCV during the first week of training. All indices of iron status (hemoglobin, PCV, 22 FEP, transferrin saturation, serum iron) returned to initial values between the first and eighth week of the season except for TIBC, which was significantly greater than preseason values. Results suggested that the young women's recovery from sports anemia could impose a demand on their body iron reserves. Although serum iron and percent transferrin saturation returned to their initial values within one week after training, the fact that TIBC peaked at that time suggested that the runners' bodies were actively attempting to restore iron reserves that had been diminished during training. Several investigations on long distance runners were conducted to analyze the levels of iron status (7,9,10). Clement et. al. (9) reported that 29% of the men and 82% of the women long distance runners had plasma ferritin concentrations at risk for iron deficiency. The male runners had an adequate dietary intake of iron (mean intake was 18.5 mg per day). In contrast, the female runners had an inadequate intake (mean intake was 12.5 mg per day) which may have contributed to the high percentage of those subjects having low plasma ferritins. Kilbar (11) investigated the effects of seven weeks of training on the serum iron levels in three age groups (including ages 9-31, 37-48, and 51-64 years) of relatively inactive women. Serum iron levels significantly decreased in each group indicating that there was a significant iron cost associated with physical training. However, the lack of a control group and failure to account for dietary and menstrual factors - each of which may affect serum iron levels - must be taken into account when interpreting these results. If there had been an iron cost of physical training, serum iron levels would decrease only if this cost exceeded storage iron 23 levels. Due to the high incidence of latent iron deficiency among runners, many researchers have studied possible causes of this. Ehn et. al. (10) found indications of latent iron deficiency, as measured by bone marrow iron, in all eight of the male long distance runners studied, even though each had an adequate dietary intake. Ehn suggested that the iron deficiency may be caused by a disturbance in iron absorption among these individuals due to the running. Iron absorption was measured with the aid of 59Fe labeled ferrous sulphate, 59Fe labeled hemoglobin, and a whole body counter in this group of elite runners and compared to eight nonrunning control subjects having similar iron status. The mean absorption of the ferrous sulfate was 16.4% in the runners, which was much lower than that of the control group which was 30.0%. The difference in absorption of hemoglobin iron was less pronounced; the runners absorbed 4.3% less than the controls. Plasma iron clearance was 20% greater in the runners than controls, indicating a greater iron loss. This was measured by the rate of disappearance of an intravenous injection of trace amounts of radioiron 59FeCl3 in the plasma. Labeled iron incorporation into red blood cells was slightly greater in control subjects, indicating a higher rate of erythropoiesis. Another contributing factor for a lower iron balance could be the result of excess sweating in conjunction with running. In one study (48), Paulev measured an additional iron sweat loss of 0.4 - 1.0 mg. per day increasing normal daily losses of 1 mg. Veller et. al. (49) found no relationship between the iron concentration of cell-rich or cell-free sweat and hematological indices or serum iron levels, with 24 the exception of a positive correlation between the iron concentration of cell-free sweat and the serum iron values after sweat collection. These findings indicate that the iron lost through sweating could not be controlled by an individual with iron deficiency. In another study (50), similar results were reported to occur. No significant differences were noted in the iron content of cell-free sweat between the normal and the iron-deficient groups. Normal subjects had a mean iron content of 1.2 mg. per liter of sweat. Hot climate and/or exercise could increase sweat loss by 2 to 11 liters, therefore, increasing the range of total amounts of iron loss to 2.5 - 13 mg. per day. In the event of a hemolytic state, as indicated by sports anemia, some of the hemolysis would occur intravascularly resulting in an elevation of free hemoglobin. This hemoglobin would combine with haptoglobin to form a complex molecule, and then be removed from the circulation by the reticuloendothelial system and eventually be used again in the synthesis of new iron-containing compounds, thus conserving iron. Circulating free-haptoglobin levels, therfore, would also decrease. If the hemolysis continued and/or exceeded the hemoglobin-haptoglobin binding capacity, excess hemoglobin would appear in the urine, after the reabsorbing capacity of the kidney tubules has been exceeded, producing hemoglobinuria. However, there could be varying degrees of exercise-induced hemolysis before the hemoglobin-haptoglobin binding capacity was exceeded (51). Hemoglobinuria has been the focus of many studies on exercise-induced hemolysis. Nine out of 50 males completing a marathon showed gross or microscopic hematuria; all abnormalities cleared up within 48 hours 25 (52). Gross hematuria has been previously reported to be an infrequent occurrence after running. In contrast, recent investigations have described up to 21 such cases among the participants of a long distance running event in which no intrinsic urinary tract cause was found (53). One investigator (10) suggested that this hemoglobinuria and hematuria could account for the iron loss producing the latent iron deficiency found in runners. Low serum haptoglobin values - indicating increased intravascular hemolysis - has been commonly reported among athletes (8,10,47,53,54,55). In a more recent hypothesis (56), the investigator suggested that this increased level of hemolysis, producing an increase in the hemoglobin-haptoglobin complex, caused a shift in red cell catabolism from the reticuloendothelial system to the hepatocytes. This would be a reasonable explanation of a reduced content of hemosiderin in the bone marrow cells and low serum ferritin levels because these values reflect the content of iron in the reticuloendothelial system. Thus, it was concluded that runners "anemia" was not caused by an increased iron loss as detected by low iron stores. No obvious explanation could be given for the single divergent laboratory values which indicated low iron stores. The low serum ferritin and low bone marrow hemosiderin values often found in athletes indicate the need for further studies on iron kinetics in this group to clearly explain these differences (55). IRON DEFICIENCY 2 EFFECTS ON PERFORMANCE Three fundamentally different questions are relevant to the potential biological effects of iron deficiency anemia. First, to an athlete the important question is related to maximum performance 26 capacity. Second, to the worker whose survival depends on his/her job performance, the daily work productivity is often a crucial factor, particularly in developing countries. A third question is related to a person's general sense of well-being or vitality, though it remains to be determined to what extent this is affected by iron deficiency anemia (57). The impact of iron deficiency on endurance athletes is illustrated by the fact that a person with a hemoglobin concentration of 12 g/dl can carry only 75% of the oxygen that a person with 16 g/dl can carry with equal red blood cell volumes (36). The total body oxygen needs are the summation of all the individual requirements of tissues and organs and vary as a consequence of functional changes occurring in everyday life. Under normal conditions, physical activity is the most important factor in determining total oxygen requirements, at least in quantitative terms. This is so because it induces significant increments in the metabolic rate of skeletal muscle and to a lesser degree in that of myocardium (58). The lower levels of hemoglobin in anemia impair oxygen delivery to the tissues to a degree which depends on the severity of the anemia and on the energy demands. Therefore, a sedentary person or someone engaged in light and intermittent work may experience no symptoms of anemia with a moderate hemoglobin deficit and may function in essentially the same manner as a normal individual; whereas someone with more severe anemia and/or who engages in physically demanding activities must resort to various physiological compensatory mechanisms. This type of individual may be forced to reduce his/her work output or modify his/her work pattern if the physiological compensation proves to be insufficient (57). In several studies (57,59,60,61,62,63,64), 27 investigators have shown that physical working capacity in different populations, measured as oxygen consumption, significantly decreases in iron deficiency anemia and improves when hemoglobin levels reach normal values. Edgerton et. al. (64) measured selected parameters related to work tolerance in 31 adult subjects with hemoglobin from 2.5 to 14 g per dl. Work tolerance was closely related to hemoglobin concentration regardless of the adequacy of storage iron level. The data strongly suggested that the decrement in work performance capacity in iron-deficient and anemic subjects was in a large part, a reflection of the level of anemia rather than other nonhemoglobin- related biochemical changes that could accompany prolonged iron deficiency anemia. Iron deficiency of short duration and of moderate degree can be associated with hemoglobin concentration in the normal range. Therefore, there may be an overlap of iron-deficient and non-deficient individuals with normal and minimally decreased hemoglobin levels. Several studies (31,47,61,65,66,67) have demonstrated that iron deficient, but not anemic subjects, could benefit from iron treatment, producing an increase in physical work capacity without significant change in hemoglobin level. Administering an iron supplement to a deficient subject reduces the stress of physical activity more than can be expected by the improvement in oxygen carrying capacity of blood alone (68). Iron may be incorporated into tissue in a manner functionally beneficial to the extent that work tolerance can be improved (67). In the past, the investigation of iron deficiency anemia has been focused on hemoglobin. However, it has been becoming quite clear that 28 the symptomatology of iron deficiency anemia reflects a complex systemic condition involving almost all cells in the body (69). Symptomatic improvement in response to iron treatment before a significant increase in hemoglobin concentration would indicate a possibility of an early repair of tissue heme protein deficiency (70). Several experiments in rats (29,61,71,72,73) demonstrated that iron deficiency was associated with a decreased concentration of cytochrome c oxidase in various tissues, a decrease in concentration of muscle myoglobin, and in certain instances a diminshed activity of iron- containing enzymes such as succinic dehydrogenase and aconitase. Severe iron deficiency caused a general decrease in the concentration or activities of iron-containing components of the electron transport chain in mitochondria isolated from rat skeletal muscles. It also caused a loss of mitochondrial protein from that tissue (72). Because of these reductions, the iron deficiency produced a decrease in skeletal muscle capacity for aerobic metabolism, and, by this mechanism, an increase in susceptibility to fatigue (73). It has been suggested (66) that the dramatic improvement in symptoms that often occurs prior to a significant rise in hemoglobin concentration during iron therapy was due to early repair of tissue iron deficiency. In all muscle tissue examined (of iron-deficient Sprague-Dawley rats) the 59Fe preferentially entered the mitochondria (74). The enhanced mitochondrial uptake of iron, prior to any detectable changes in the hemoglobin level, in experimental animals could be indicative of nonhemoglobin related biochemical changes and/or decrements in work capacity (74). In one experiment (75), rats were raised on a severely iron- 29 deficient diet and then exchange-transfused to a normal hemoglobin level. These animals demonstrated an impaired work capacity (as measured by length of treadmill running time) accompanied by an increased lactate production, as compared to control animals on a chow diet. Given parenteral iron, the deficient animals regained a "normal" running ability within 3 to 4 days. Concentrations of the cytochrome pigments and myoglobins and rates of oxidative phosphorylation with pyruvate-malate, succinate, and alpha- glycerophosphate as substrates, were all reduced in mitochondrial preparations from skeletal muscles of iron-deficient rats. Only the rate of phosphorylation with alpha-glycerophosphate as substrate increased significantly and in parallel with the recovery of work performance of the deficient rats treated with iron. The iron deficiency apparently caused a depletion in the iron-containing mitochondrial enzyme, alpha-glycerophospate oxidase, which in turn impaired glycolysis. This resulted in an excess lactate formation, which at high levels leads to cessation of physical performance observed in iron deficiency (29). Iron deficiency alone could cause an elevation of resting blood lactate levels in human beings and rats. Findings have suggested that iron deficiency with or without anemia has caused an increase in tissue anaerobic glycolysis, resulting in an accumulation of lactate (77). Blood lactate concentrations following a period of exercise provide an estimate of the amount of energy obtained from anaerobic sources. It may be used to assess the relative levels of skeletal muscle hypoxia incurred during these activities (78). Post exercise lactate concentrations also appear to be directly related to the 30 degree of anemia. Lower lactate concentrations have been observed after exercise in the subjects with the highest hemoglobin concentration, in spite of the fact that they worked longer and at higher work loads than those subjects with hemoglobin concentrations below 13 g. per dl. (79). Iron-deficient rats with adequate hemoglobin levels displayed muscle activity associated with a higher blood lactate concentration than that observed in iron-repleted animals. The accumulation of lactate appears to be the result of excessive production, as lactate clearance from the blood has been shown to be unaffected (76). Two weeks of iron therapy in minimally iron-deficient women athletes resulted in lower lactate levels at the end of an exhaustive exercise (47). Aerobic metabolic capacity can be restored by iron treatment. The lower post-exercise lactate and higher partial pressure of carbon dioxide found after iron treatment may be caused by such improvements of mitochondrial oxidative phosphorylation, changing the relative metabolism from anaerobic to aerobic (77). In a brief hard exercise, maximal oxygen consumption (VO2 Max) would decrease in a subject with iron deficiency. Additionally, the ability to perform prolonged submaximal endurance exercise would also be diminished. Perkkio et. al. (80) had shown that the depressed VO2 Max in rats with severe iron deficiency anemia was virtually corrected by red cell transfusion whereas endurance remained impaired. The results suggested that the concentration of hemoglobin was a major determinant of V0 Max, whereas mitochondrial oxidative capacity was 2 more likely to be the limiting factor in endurance capacity. Severely impaired duration of performance in a submaximal exercise (endurance) 31 was related primarily to a decreased muscle capacity for oxygen utilization (due to a decrease in iron-containing compounds) rather than dimished oxygen delivery (due to a lower hemoglobin concentration) in the blood. This study indicated that endurance exercise had become substantially impaired even under conditions of moderate iron deficiency where VO2 Max was virtually uneffected. Iaon NUTRITURE DIETARY SELECTION The average U.S. dietary intake, reported forty years ago, indicates that dietary intake supplies 14 to 20 mg. of iron per day in man. There is some evidence that iron intakes are falling as modern food handling processes reduce the opportunities for contamination (19). The typical western diets provide approximately 6 mg. total iron per 1000 kilocalories (32). A decrease in total caloric intake and a reduction in use of iron pots may also contribute to lower iron intake today. The more important change, however, may be related to the availability of iron to be absorbed from food (28). The adolescent's nutritional status has been of increasing concern in the past two decades. The data on nutriture of the adolescent population has shown that intakes of iron were lower than intakes of any other nutrient (19). Earlier studies (4) have established that adolescent girls have the highest prevalence of unsatisfactory dietary intake of iron in all age groups surveyed. The average intake among girls has been reported in many studies as 10 to 12 mg. per day. In the Ten State Nutritional survey (3), 80% of females of all age groups had iron intakes below the recommended daily allowance (RDA) of 18 mg. Approximately three-fourths of the boys, 12 to 16 years of age, 32 consumed less than the RDA of 18 mg. of iron per day, while more than one-third of the boys, 10 to 11 years of age, consumed less than the RDA of 10 mg. of iron. According to the U.S. Department of Health, Education, and Welfare dietary intake findings from the Health and Nutritional Examination survey of 1971 - 1974 (5), mean intakes of dietary iron were 73.2% and 57.7% of the RDA for male and female adolescents, 10 - 14 years, respectively. According to the U.S. Department of Agriculture survey of 1977 - 1978 (6), 36% of adolescent males and 64% of females had less than 70% the RDA of 18 mg. dietary intake of iron per day. The Recommended Dietary Allowances have been set to afford a sufficient margin above the physiological requirement to cover variation among essentially all individuals in the general population (81). That this objective with respect to iron has been rarely met by intake of ordinary foods, at any age during adolescence, has been a well accepted fact (4). Adolescents tend to consume a large portion of their daily energy intake as snacks. In the latest household food consumption survey (U.S.D.A.,I980) 9 to 11 year old males and females consumed up to 12.7% and 12.0% of the total daily energy, respectively, from snacks (82). According to the Ten State Nutrition Survey, both male and female teens obtained a substancial proportion of their recommended kilocaloric intake - approximately 23% - from between-meal snacks. The mean nutrient intake per 100 kilocalories from between-meal snacks did not meet the RDA for iron, contributing only 12% of their total iron intake (3). In their quest for independence, adolescents spend more time away from the home and consume more of their meals and snacks outside of the home. Fast-food restaurants and vending 33 machines are popular choices because they provide inexpensive foods in a short period of time. The iron density of many of these foods (except for the red meat hamburgers) in relation to the kilocaloric intake, is low in comparison with the iron requirements of teenagers. Certain meal combinations, from vending machines and fast food restaurants, are excessive in energy when compared with the amount of nutrients provided. These foods have been acceptable nutritionally when consumed judiciously and as part of a well-balanced diet. When these snack foods have become the mainstay of the diet, however, there has been cause for concern and some form of iron supplementation or enrichment would then be suggested (40). Elite athletes' diets may have important effects on their responses to their endurance training programs (9). Limited information on the dietary patterns, in relation to nutrient intake, in runners is available. Blair et. al. (83), compared the nutrient intake of middle-aged male and female (35 - 59 yrs.) runners and controls, of the same age and gender. The runners were leaner and had higher energy intake; 40 to 60% more kilocalories per kilogram body weight than the heavier sedentary controls. The increased energy intake of the runners was associated with higher intakes of fats and carbohydrates than by the controls. The runners consumed relatively less protein per 1000 kcal, although the absolute amount of protein in their daily diet was similar to that of the controls. Clement and Asmundson studied (9) the nutritional intake in 52 middle-distance and distance runners, and reported adequate levels of mean energy intake for both males and females. The women had a mean iron intake of only 69% of the recommended daily intake of 18 mg per day. The men had a 34 mean intake which was adequate at 18.5 mg. per day; the recommended daily intake is 10 mg. per day. These data support the hypothesis that runners eat typical American diets, with the only difference being a higher energy intake than nonrunners to cover the increased energy expenditure associated with the participation in the sport (83). ‘IRQN SUPPLEMENTATION Several investigators (12.13.14.15) have reported the effectiveness of pr0phylactic supplementation of iron in athletic and sedentary children, adult men and women, both anemic and normal. The dose of iron given in the form of ferrous sulphate or ferrous fumarate has varied from 5 mg. to 400 mg. a day and the duration of supplementation from one month to three years. The results have been variable depending upon the dose of iron used, duration of supplementation, and iron status of the population studied (22). In two studies (56,71), where hematological investigations and iron status measurements were done on middle and long-distance runners, no differences were found in these measurements between the athletes taking iron supplements and those that did not. Cooter and Mowbroy (12) investigated the effects of iron supplementation on serum iron, TIBC, hemoglobin, and MCHC in female college basketball players divided into two groups; one received a multivitamin with iron and the other received a multivitamin without iron. No significant changes were observed in any of the experimental variables, including hematological parameters, blood iron indices, and performance capability, over the duration of the four month study. In another study (13), College athletes randomly divided into an iron 35 supplemented group and a control group were followed during their training and competitive seasons. No significant changes were observed in any of the hematological indices, serum iron, or percent saturation of transferrin measured in each group throughout the nine week season. Contrary to the previously mentioned investigations, results from one study (14) showed significant difference between the supplemented and control groups. In this study, higher dosed supplements of 250 - 300 mg. of iron per day, along with vitamin C (to increase the availability of the iron from the supplement) were given to female athletes. The group receiving iron supplementation showed significantly greater hemoglobin levels, with a mean increase of 1.0 g/dl, than the placebo-supplemented group. TWo investigators (13,15) compared the performance capability of an iron supplemented group with that of a nonsupplemented group in association with their state of iron nutriture. No significant differences were found in the hematological and iron status between the groups, but measures of physical work capacity and performance capability were greater in the iron-supplemented group. That no significant difference was found between groups has been consistant with the statement that athletic training differs primarily in the extra energy requirement and the needs for hypertrophy of the muscle. Improvement in performance capability may be the only indication that: prophylactic iron supplementation during training would be needed by top athletes (13). Suboptimal hemoglobin (which designates a hemoglobin concentration that is lower than could be considered optimal for oxygen transport purposes) is not a major concern to the non-athletic population. 36 Therefore, relatively little is known about how to treat it. Researchers are not even sure that hemoglobin concentration in the normal range can be increased with standard noninvasive techniques. It seems apparent that a larger scale clinical trial of iron therapy in athletes with suboptimal hemoglobin is needed to answer some of these questions (85). It would be premature to conclude from studies conducted thus far that iron supplementation should be given to athletes as a way of improving performance capabilities. More definitive research must be done in this area before such a recommendation can be made. At present, the prudent recommendation regarding prevention of sports anemia and suboptimal hemoglobin seems to be: to ensure that athletes ingest a diet that provides at least the RDA of iron, protein, vitamin C, vitamin 812, and folic acid; to screen athletes routinely for iron deficiency and hemoglobin concentration; and to prescribe iron and/or other dietary supplements for athletes who manifest low hemoglobin levels or iron deficiency (85). IRON BIOAVAILABILITY IRQN_ABSORPTION The average American diet contains over five times the total amount of iron needed to maintain iron balance, but only a small percent of this is absorbed (86). The nutritive iron value of special foods or categories of food should be based on at least 3 considerations: concentration of iron, form of the iron in terms of its availability, and influence on iron availability by foods ingested simultaneously (87). Bioavailability is a measurement of the potential use of a mineral, nutrient or drug by an organism. Since 37 the conversion of the nutrient to an active species is essential, bioavailability thus encompasses more than just intestinal absorption. Factors which influence the measurement of bioavailability of iron from different meal combinations include solubility, pH, chemical form of iron, oxidoreductive activity of iron, concentration of iron in the meal, capacity of iron to form complexes (with inhibitors and promoters), food digestibility, food processing conditions, and nutrient interactions. Although this list is far from complete, it can readily be seen that these factors are multifactorial and interdependent. Therefore, the control of all but a single variable is difficult in food systems (86). Absorption of dietary iron is regulated in the normal individual by physiological need (iron status) and will vary from meal to meal as modified by the composition of each meal (88). This regulation of absorption is closely tied to the level of iron stores (absorption increases with reduced stores and decreases as stores enlarge) and the rate of erythropoiesis (30). With respect to its availability, food iron can be divided into two main parts: heme and nonheme iron, each of them forming a different pool of iron (86). The availability of heme iron, which is present as hemoglobin and myoglobin in animal tissue, is high in comparison to nonheme iron, averaging 15% for iron- replete men to 35% absorbed in those lacking iron stores (89). Although heme iron constitutes only 5 to 10% of dietary iron ingested in a Western diet, it accounts for nearly 30 to 40% of the iron absorbed from the diet because of the high assimilation of heme iron by the mucosal cells (90). This increased availability is due to both the physiochemical nature of heme iron and its mode of absorption 38 (89). Heme iron is more soluble in the neutral conditions of the small intestine than in the acid environment of the stomach. It is thought to be absorbed directly into the intestinal mucosa as the intact iron porphyrin complex. The enzyme, mucosal heme oxygenase, splits the iron from the porphyrin ring in the intestinal mucosal cell (23). The iron then enters the same storage or transit pathways as the nonheme iron fraction. However, in another hypothesis (91), a different pathway for the hemoglobin iron absorption is suggested. During meat digestion, heme is split from the globin in the stomach. After further digestion in the intestine, the heme ring is cleaved with the released iron, becoming loosely bound to one of the many fragments derived from the digestion of the meat proteins. This iron may then enter the intestinal mucosa as inorganic iron or may even be absorbed as an amino acid or small peptide chelate. Wheby et. al. (92) studied the hemoglobin iron absorption kinetics in iron-deficient dogs. He found very little free iron acculmulation in the mucosal cell during the absorption of 59Fe labeled hemoglobin, suggesting the absence of a feed back inhibition on the heme oxygenase. The mucosal 59 Fe heme had accumulated over time while the free 59Fe did not, indicating rapid transport of the 59Fe split from heme. These results suggested that the rate limiting step in absortion of hemoglobin iron in iron-deficient dogs was the splitting of iron from heme in the mucosa. This control mechanism could explain the fact that iron-deficient humans absorb more iron from a dose of elemental iron than from the same amount of iron given as hemoglobin. Except for the facilitating effect of meat on the absorption of heme iron, the type of meal has little influence on its absorption 39 because the iron remains within the porphyrin complex until it is absorbed by the mucosal cell (93). From studies involving different dose levels of heme iron, it appears that the bioavailability of heme iron in meals containing meat has a maximum absorption of about 25% while the bioavailability of heme iron given without meat or liver has a maximum absorption of only 10% (16). Using in-vitro studies with simulated intestinal loops, Hazell et. al. (94) found that when meat or hemoglobin was ingested with a large amount of protein, the end products were low molecular weight, nonheme compounds which were easily absorbed. Hazell also found that hemoglobin remained as the end product to be absorbed only when it was digested alone. Thus, hemoglobin was absorbed intact when digested alone, but absorbed as a nonheme iron complex when digested with meat in simulated digestion studies. More in-vitro studies need to be conducted to explain the differences in digestion and absorption of iron from hemoglobin or meat. Thus, the chemical nature of the heme-iron absorption phenomenon is still not clearly understood (89). Iron from foods such as vegetables, fruits, cereals, eggs, dairy products, as well as nonheme iron of meats, poultry, and fish and from soluble iron supplements all form a common nonheme iron pool - representing the largest fraction of iron in the meal (23). Assimilation depends on the extent to which iron remains soluable within the lumen of the upper small intestinal tract - largely affected by the general composition of the meal. DIETARY FACTORS INFLUENCING BIOAVAILABILITY During the passage of food from stomach to duodenum, the pH increases from 1.5 to 7.0 due to duodenal secretions. As a result, 40 most ferric iron is precipatated as ferric hydroxide or phosphate unless this is prevented by the presence of chelating agents. Ferrous iron, however, is not so readily precipated in the duodenum, and a significant proportion of it is still soluble even at a pH of 7.0 and hence is better absorbed than the ferric iron. It is the iron which is present in the duodenum and upper parts of the jejunum in a soluble form that is available for absorption (22). While increasing amounts of inorganic iron are absorbed from larger test doses, the percent absorbed progressively decreases. This indicates that intraluminal iron molecules compete with each other for either: binding to intraluminal substances which facilitate absorption; passage through absorptive pores in intestinal mucosal cells; or a combination of these possibilities (95). The acceptor sites on the brush-border surface compete for iron with the ligands present in the gut lumen. Some ligands, mainly derived from foods, promote iron absorption by keeping it in solution and others inhibit absorption by precipatating iron. The results depend upon the balance of these opposing factors (22). Therefore, it is not the biological form of iron in the food, but rather the composite effect of the inhibitors or enhancers in a complete meal that determines nonheme iron availability (96). Certain compounds such as organic acids, sugars, amino acids, dicarboxylic acids and hydroxy acids, normally present in food, can form chelates with ferric iron to keep it in solution and prevent its precipitation at the neutral or alkaline pH in the duodenum. These compounds are considered to be enhancing factors for iron absorption (22). Contrary to these agents, compounds such as calcium-phosphates, phytates, and tannins can precipitate iron at the neutral pH, and 41 therefore, are considered inhibiting factors (88). Iron can be absorbed without chelating agents, but only at low pH. Besides maintaining iron in available form, the chelating agents also provide a specific transport mechanism for iron chelates similar to heme (97). Ascorbate acts as a ligand for the common nonheme iron pool. Enhancing effects of ascorbate are directly proportional to the quantity added to the meal. This vitamin can also reverse the inhibitory effects of tannins, calcium, and phosphates on nonheme iron absorption (96). The effect of ascorbate depends upon its presence in the meal and is unrelated to the ascorbate status of the individual (98). Cook et. al. (90) reported that vitamin C had negligible effects on iron stores when large quantities of ascorbate were included in subjects' diets for up to two years. There was a fivefold enhancement of iron absorption from foods when 1000 mg. ascorbate supplements were taken with the meal. If this enhancement had been maintained for 2 years in a normal adult male, with a basal absorption of 1 mg. iron daily, the body iron stores should have increased to greater than 3 grams. These results were not observed among the subjects. Apparently, the regulatory mechanism controlling body iron reserves overrides any pronounced alterations in food iron availability. In several studies (93.97.99), the presence of animal flesh in the diet has been shown to result in an increase in the absorption of nonheme iron, possibly by stimulating the digestion of food or counteracting the inhibiting factors on iron uptake. The absorption- promoting effect of meat seems to be of the same magnitude for both kinds of iron. This fact suggests that there is a common mechanism of 42 action of meat on the absorption of both heme and non-heme iron (16). The animal tissues; beef, pork, lamb, liver, chicken, and fish each increase iron absorption to a different degree. Because dairy and animal products such as milk, cheese and eggs inhibit absorption, the enhancing effect of meat on nonheme iron absorption may be due to some component of animal tissue unrelated to the protein content. Alternatively, it may relate to the amino acid composition of the administered protein (100). The mechanism of animal protein enhancement on dietary iron still remains unanswered (87). The enhancing effects of ascorbate and meat are directly proportional to the quantity present in a meal - but the combination of both in facilitating iron absorption is not additive (101). This suggests that animal tissue and ascorbate enhance iron absorption by the same mechanism (98). Sub-optimal iron nutriture may become an increasing concern if the consumption of plant foods continues to rise (33). Iron absorption from grains and vegetables is markedly inhibited by the concommitant ingestion of dietary components such as dietary fiber and phytic and tannic acids present in plants (7,88,102). Fiber binds minerals in the digestive tract, possibly rendering them unavailable for absorption in the body. Absorption of minerals might also be decreased because of the diluting effects from the extra water taken up with the fiber or because of the faster transit (due to the fiber) through the intestines (103). Iron binds to tannates and phytates to form complexes which precipate, making them unable to be absorbed (16). Calcium and phosphorous individually, cause slight inhibition on nonheme iron absorption, but not as effectively as calcium- 43 phosphate complexes. The phosphate precipitates more efficiently by iron with calcium present especially at the higher pH of the small intestine (104). Other inhibitors of iron absorption include; the chelating agent, EDTA, used to prevent metal oxidation in many foods, tannins found in tea, polyphenolics found in coffee, phosphoproteins found in eggs and cows' milk, and lectins found in soy proteins (88). Even though the percent absorbed of nonheme iron is considerably less than heme iron - due to inhibiting actions of many dietary components - the quantity of nonheme iron in the diet is many fold above that of heme iron, therefore, the major contribution of available iron is made by nonheme (17). MEASUREMENTS OE DIETARY BIOAVAILABILITY 95.139! Various methods have been used to study the dietary bioavailability of iron in man. The chemical balance technique is the only method that directly measures iron absorption from the whole diet. This method, however, is insensitive, imprecise, and time consuming, and it gives no information on iron absorption from different meals (16). Other in-vivo techniques measure: the increase in serum iron concentration following administration of an oral dose of iron from different iron compounds; changes in body iron stores as an indicator of iron utilization vs. iron availability from foods; and the rate of hemoglobin repletion in iron-deficient animals after eating various dietary sources of iron (105). The introduction of radioisotopes has made it possible to label single food items biosynthetically with radioiron (16). In recent studies (101,106), investigators found that there is an almost complete isotopic exchange between the non-heme iron compunds in the 44 foods and the added inorganic radioiron tracer. The absorption of such a tracer mixed into the diet can thus be expected to give a true measure of the total absorption of non-heme iron from a diet. The absorption of radioiron labeled hemoglobin mixed into the diet may give a fairly true measure of the total absorption of heme iron from the diet. The absorption of food iron might thus be considered to take place from two pools - one heme iron and one non-heme iron pool (101). After administration of a radioiron-labeled food or meal, blood samples are taken over a period of time to measure the amount of radioiron in either whole blood or hemoglobin. Instead of taking blood samples, one can use whole-body counting to calculate the percent of retention vs. time after the radiolabeled food or meal is eaten (105). This method has greatly facilitated iron nutrition studies of various kinds. Further studies are necessary - especially on the exchangeability of nonheme iron in a wider variety of foods involving different preparation techniques - to permit application of the method in studies involving different main dietary iron sources: (101,105). ESTIMATION OF AVAILABLE DIETARY IRON Because iron absorption varies so markedly according to the diet, recommended dietary allowances merely provide rough guidelines. It is possible, however, to develop iron deficiency while ingesting large amounts of iron-rich foods in which the iron is poorly absorbed, or conversely, to escape deficiency despite ingesting far less than the recommended amount but in forms that are particularly well absorbed (1). The current Recommended Dietary Allowance for food iron intake has been established assuming an absorption of 10% of the total 45 amount present in the daily intake (82). However, this amount can rarely be achieved with the ordinary foods available (17). The 1980 Food and Nutrition Board (81) has recognized the importance of iron bioavailability; the critical issue relating diet and iron status is not the total amount of iron ingested but, rather the amount of iron available for absorption. Research on iron absorption has extended further than that on the absorption of other trace minerals, and thus iron is the first trace mineral for which a model to estimate bioavailability has been developed (18). Accumulating evidence demonstrates that the amount of iron potentially available from foods depends not only upon the amount supplied but the nature of that iron and the composition of the meal. The total iron content of the diet is thus a relatively poor indicator of the adequacy of the diet with regards to this mineral. Although numerous questions remain to be answered about iron bioavailability and iron needs, sufficient information is now at hand so that better estimates of iron need in relation to diet can be made. Such information should be used in the development of diets and in making dietary recommendations (17). Monsen (17) developed a model which categorizes the meal as high, medium, or low availability of the nonheme iron pool with a certain percentage associated with each, indicating the amount of iron able to be absorbed. These categories are based on the amount of ascorbate and meat present and the level of iron stores in the individual. In order to derive the percent of absorbable iron present in a meal one must: calculate the iron content of individual meals and snacks; calculate the amount of heme iron in these meal units and the amount 46 of bioavailable heme iron; calculate the amount of non-heme iron in these meal units; assume absorption of the non-heme iron to be at a low level unless there is concomitant ingestion of enhancing factors; sum up the enhancing factors and calculate the amount of bioavailable nonheme iron one would anticipate; and finally sum up the bioavailable heme and non-heme iron to arrive at the total amount of iron absorbed (18). Appendix 8 illustrates this model. Several assumptions have been made with this model which restricts its application. All types of heme iron are assumed to be equivalent in percentage of the iron found in the heme complex of the animal tissue. Certain forms of nonheme iron are not entirely exchangeable with the nonheme pool - particularly fortification iron in compounds of low availability - which should not be included into the calculations. Inhibitory factors are not taken into account. Iron status of the individual is split into three separate categories with one specific percentage associated with each group. All of these assumptions decrease the credibility of the model in calculating a specific amount of absorbable iron in the meal (17). In that the goal is to increase the quality of the diet by increasing the quantity of bioavailable iron, it makes no difference in comparative analysis of diets which level of body iron stores one utilizes for calculations. It is suggested, however, that the individual with 500 mg. iron stores be selected as a basis for comparison. For the individual with zero iron stores, one would anticipate approximately twice as much iron to be bioavailable as would be calculated for the reference individual with 500 mg. iron stores. One can, therefore, use this model to gage the quality of the diet in facilitating iron absorption. In the 47 effort to improve the iron nutriture of an individual one can; increase the content of factors which will enhance the absorption of nonheme iron, decrease the ingestion of inhibiting agents, and improve food choice to increase ingestion of dietary iron and improve its bioavailability (18). METHODS SUBJECTS: Subjects for this investigation were part of an extensive study on fitness and performance conducted by the Youth Sports Institute, through Michigan State University. The sample included; subjects evaluated in Phase II of the study, August, 1983, consisting of 23 male and 15 female runners and 9 male and 7 female nonrunners, and in Phase III of the study, September, 1984, consisting of 21 male and 13 female runners and 11 male and 10 female nonrunners. Nineteen male runners, 10 female runners, 10 male controls, and 6 female controls participated in both Phase II and Phase III. One male runner and 1 female runner in Phase II had stopped running and became control subjects in Phase III. All except 2 subjects were caucasian (the other 2 subjects were hispanic), ranging in age from 8.5 to 16.9 years. The runners were selected if they were one of the top three finishers in their age group in road races in the state of Michigan during the year of testing or during the previous year, represented by Phase I of the study. The controls were selected to match the ages and heights of the runners for the first year, Phase I, of the study - Fall, 1982. The control subjects were normally active, but did not undergo heavy training for running road races. The control subjects were selected from among the participants of an ongoing motor 48 49 performance study conducted at Michigan State University. Due to differences in growth rate and discontinuation of some subjects, pair- matched controls could not be maintained throughout the course of the study. Hematological parameters were not measured in blood samples obtained from subjects in Phase I of the study. Hence, data from the other tests (treadmill and dietary records) conducted in Phase I were not included in this study. Written informed consent was obtained from all subjects and their parents prior to testing. The study was approved by the Committee on the Use of Human Subjects, Michigan State University. BLQQQIANALYSIS: The subjects participated in a full day of testing at the Center for the Study of Human Performance, beginning with blood collection at 7:30 a.m. Blood was drawn with stasis from the anticubital vein of each subject after a 12-hour fast. Five milliliters of blood were drawn into vacutainers containing disodium edetic acid anticoagulant for determinations of hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), and red blood cell count (RBC). These measurements were determined by an eletronic coulter counter. This and the other determinations were done at the Lansing Clinical Laboratory, Lansing, MI. Seven milliliters of blood were collected in heparinized vacutainers for mineral analysis. The tubes were inverted to prevent clotting and cooled to 4°C immediately, by being placed in a refrigerator (4°C) at the testing site. Three to seven hours later, the heparinized tubes of blood were spun down - at 2400 RPM (1300 g) for 15 minutes 50 at 5° in an IEC model PR-6000 centrifuge1 - to harvest the plasma. Plasma samples were frozen in polystyrene storage tubes for later analysis of iron(107). Plasma iron was determined by atomic absorption spectrophotometry using an Instrumentation Laboratory (model 951) Spectrophotometer2 following the method of Olson and Hamlin (108). Duplicate aliquots of 0.5 milliliters plasma from each sample were deproteinized with trichloracetic acid (Mallinkrodt3) and incubated in a water bath at 90°C for 15 minutes. Immediately afterwards, the samples were refrigerated at 4°C to cool down, then centrifuged at 2000 RPM (1080 g.) for 15 minutes. Supernatants were harvested, and used undiluted for iron determinations which were done the same day, and directly after preparation of the standard curve for iron. Samples were read against aqueous standards prepared from iron stock solution (JT Baker Chemical 00.4). Any duplicate samples which were less than 90% of each other were discarded and determination of iron was made on freshly prepared samples. Due to the limited amount of plasma, repeat analyses to obtain lower percentage of error was not possible. ANTHROPOMETRIC MEASUREMENTS: Heights and weights were measured on each child during the day of testing. Heights were measured to the nearest gDamon/IEC Division. Needham Hts., MA. 3Instrumentation Laboratory, Inc. Wilmington, MA. 4Mallinkrodt, Inc. Paris, KY. J.T. Baker Chemical Co. Phillipsburg, NY. 51 millimeter with the subjects, without shoes, eyes straight ahead, with heels and shoulders touching the wall. Measurements were made with a free-standing anthropometer. Weights were measured to the nearest gram with a medical beam balance, with the subjects in bare feet and bathing suits. All subjects were weighed on the same balance. A Tanner rating (109) on each child was evaluated by a physician in Phase II, 1983. Hand and wrist x-rays were taken on some of the subjects during the spring of 1984; after Phase II and before Phase III of testing. These x-rays were used to determine the skeletal age of each subject. This was done by matching his/her x-ray to a standardized atlas of roentgenograms compiled by Greulich and Pyle (110). Recorded weights and heights in Phase III were plotted per age of each subject on the NCHS growth charts (111) for boys and girls. Relative weights and heights were calculated by dividing the actual measurements by the measurements for the 50th percentile of their age/sex group times 100. The height measurements of each subject, for each phase of the study in which they participated, (four years for those subjects who participated since the first year and two or three years for those who joined the study the following years) were plotted individually on the NCHS growth chart (111) to determine the stage of growth of each individual. The three stages of growth were: the "pre" category which included those subjects who had not reached their growth spurt, gradually increasing in height at a constant rate along a growth curve percentile on the NCHS growth chart (111); the “peak“ category which included those subjects who were undergoing a 52 rapid increase in height, accompanied by all the changes associated with puberty; and the “post“ category which included those subjects who had slowed down their rate of growth, indicating near completion of puberty. To illustrate how subjects were categorized, a line representative of growth for each of the three stages is plotted in Figure 1. Phase IV data were available but not included in this study. However, the heights of each subject in Phase IV (Fall, 1985) were plotted to give a better indication of the stage of the growth spurt for each subject in Phase II and Phase III. The Tanner ratings and skeletal bone ages (of those subjects who had hand and wrist x-rays taken) were referred to, to check the accuracy of the prediction of each subject's growth category. 53 Figure 1. Growth curve lines for the 3 growth categories; pre-, peak-, and post-growth spurt, plotted on a NCHS growth chart. 54 mus ' necann # nomi- 55 PERFORMANCE CAPABILITY AND ANAEROBIC METABOLISM: A stepwise treadmill run to exhaustion was conducted to obtain the respiratory measurements; percent values of carbon dioxide and oxygen in exhaled breath and gas volume of the lungs, to determine maximum oxygen consumption. The subjects completed an intermittent treadmill running protocol of progressively more intense 3-minute work intervals followed by 3-minute resting intervals until they reached exhaustion. Treadmill speed started at 6 miles per hour (MPH) with the grade set at 0%, initially. The treadmill incline was increased by 1% and the treadmill speed was increased by 1 MPH at each subsequent work-interval level. Respiratory variables determining the maximum oxygen consumption were obtained by a modified Douglas method (112). Subjects' expired air was collected through a 2-way low-resistance (Danielss) respiratory valve into lightweight, neoprene weather balloons (113). Bags were changed every 30 seconds by an automated switching valves. The percent of carbon dioxide and oxygen contents of the air were determined immediately after collection, using an applied electrochemistry CD-38 infrared carbon dioxide analyzers and an applied electrochemistry S-3A electrochemical oxygen analyzer7, respectively. The exhaled gases were pumped through a DTM-115 dry gas 8 meter at a constant rate of 50 liters per minute for measurement of 5Model VS4S, Cambridge Instrument Co. $Van Huss Wells Automated Switching Valve. 8Applied Electrochemistry. Sunnyvale, CA. American Meter Co. (Singer). 56 gas volume. Pulmonary ventilation, oxygen uptake, and maximum oxygen uptake were calculated by the equations of Consolazio, Johnson, and Perera (112). The heart rate was determined from a 3-lead electrocardiogram and recorded continuously on a Sargent recorder to determine the maximum rate. Before work, immediately following each test level, immediately following exhaustion, and at 5, 10, and 15 minutes of recovery, fingertip, arterialized (arterialized by keeping the hand warm at 45°C) blood samples were obtained for determination of whole blood lactate. Lactate levels were determined by the Roch Lactate Analyzer 6409 (114). Immediately after the blood sample was taken, 50 microliters of this anticoagulated whole blood was added to 450 microliters of a diluting solution, (Oxford Laboratory Solution pre-prepared containing 1 gram per liter of sodium azideg) into the haemolysis tube, mixed, and then read. Subjects' lactate levels were corrected for the training effect by dividing their maximum lactate levels by their maximum oxygen consumption (50). NUTRIENT ANALYSIS: Nutrient intake was estimated from a 3-day dietary record kept by each subject prior to the day of testing. The record included 2 week- days and 1 week-end day. At the test site, subjects were interviewed (with a parent when possible) on each food record to clarify portion sizes, condiments, recipe ingredients, brands of food selected, and if any vitamin or mineral supplement was taken. Dietary records were coded and evaluated for several nutrients by the Michigan State 9Bioelectronics. Switzerland. 57 University Nutrient Data Bank (115). Nutrient intakes for each group were compared with each other as well as with the Recommended Dietary Allowances (81) for the appropriate gender and age. Subjects' nutritional intakes were evaluated to detect any significant differences in amounts of protein, animal and plant protein, and energy intake, and percent RDA for energy, protein, iron, vitamins A, C, B12, and folic acid. Other nutrients were also examined to note any extreme levels of intake. Subjects ingesting iron supplements were compared to those who did not in each group in relation to hematological and nutriture iron status. The amount of iron in supplements was added to food nutrients to arrive at total daily consumption, which was compared to the nutrient consumption levels excluding the supplements. Those individuals who took an iron suplement in one phase but not in the other were also evaluated. Iron bioavailability for each meal was calculated - according to Monsen's model (18) - for each subject at each level of iron status (0 mg., 200 mg. and 500 mg. of iron stores). Appendix B describes this method to calculate the amount of absorbable iron. This analysis was done to evaluate and compare this method with the general method of assuming an overall 10% absorption of the total amount of iron present in foods as done with estimating the percent RDA. The percent of the required 1.8 mg., as suggested by the Recommended Daily Allowance (81), absorbed was calculated using Monsen's model and compared to the percent RDA for iron. The percent of total dietary iron intake which was absorbed at 3 specified iron storage level (according to Monsen's 58 model; 0 mg, 250 mg., and 500 mg.) was compared with the percent RDA in each group. The quality of each subject's diet was assessed through use of this model by recording the amounts of absorption enhancing factors (dietary heme and ascorbic acid) present in each meal. The inhibiting factors; calcium, phosphate, tea, coffee, and dietary fiber were also recorded, though Monsen's model did not include these factors in the calculations for the amount of absorbable iron. STATISTICAL ANALYSIS: The effects of running versus developmental stage (breaking the sample into three groups; pre, peak, and post growth categories) on the dependent variables were asessed by an F ratio test, analysis of variance for a 3 by 4 multiple classification table (116). Based on the procedure by Grubbs (117), outliers were excluded in some statistical analyses. The Bonferroni t-test for unequal sample size (118) was used in the comparison of variables where appropriate. Physical characteristics, hematological measurements and dietary intake of kcal, protein, iron and vitamin C were evaluated by the Bonferroni t-test for unequal sample size (118), and compared in each of the four groups (male runners, male controls, female runners, and female controls) for both phases of the study. Hematological and plasma iron values of each group were compared to the normal median values reported for adolescents in the NHANES II survey (25). The median and range values are presented in Appendix A. Subjects with low hemoglobin and hematocrit values were individually evaluated to determine whether the low level persisted in more than one phase and 59 whether or not a specific cause of the low level could be identified. TWo of these subjects had been reclassified according to their activity patterns, from being a runner in Phase II to a control subject in Phase III. The following correlation coefficients were determined: (1) gain in height vs. change in hemoglobin and change in hematocrit from Phase II to III; (2) In Phase II, VO2 Max vs. hemoglobin and hematocrit; (3) In Phase III, VO2 Max vs. hemoglobin, hematocrit, and plasma iron; (4) In Phase III, VO2 Max vs. maximum lactate level and total lactate production (maximum lactate production - resting lactate level); (5) _In Phase III, maximum lactate level vs. hemoglobin, hematocrit and plasma iron; (6) In Phase 111, total lactate production vs. hemoglobin, hematocrit, and plasma iron. All correlations were done by the Pearson product-moment correlation coefficient (117). Hematological measurements, VO Max, and dietary iron intake were 2 compared and evaluated by the Bonferroni t-test for unequal sample size (119), between supplemental iron users and nonusers within each group. Those individuals who took supplements in one phase but not in the other were analyzed separately. A paired-data t-test (120) was used to evaluate the significance of the change in the hemoglobin, hematocrit, VO2 Max, age, height, and dietary iron intake values of when they took the supplement from when they stopped using the supplement. The amount of iron absorbed, as calculated by Monsen's Model (18), was compared between each specified iron storage level (Omg., 250 mg., and 500 mg.) versus the percent of the RDA (81) for iron in each group. Percent values for bioavailable iron were evaluated by the 60 Bonferroni t-test for unequal sample size (118) for each group to assess differences in the quality of their diets. Differences in vitamin C intake, total protein, animal protein, plant protein, and percent of dietary iron intake in the form of heme were determined for each group to compare the amount of enhancing factors in their diets. Differences in calcium, phosphate, and dietary fiber were determined for each group to compare the amount of inhibiting factors in their diets. These differences were evaluated by the Bonferoni t-test for unequal sample size (118). RESULTS CLASSIFICATION‘QE'SUBJECTS: In Phase II (Table 2), runners and controls of each gender were similar in age and height. Male runners (MR) were significantly lighter (p<0.01) than male controls (MC), and there was a trend (p<0.10) for the female runners (FR) to be lighter than the female controls (FC). The males and females of each group of runners and controls were similar in age, height and weight. However, there appear to be meaningful differences, although not detectable statistically. Although male and female runners were slightly younger than their respective controls, there were no significant differences in their ages in Phase 111 (Table 3). However, MR tended to be shorter than MC, and FR were significantly shorter than FC (p<0.05). Controls of each gender weighed significantly more (p<0.01) than runners of the same gender. When subjects were assesed for their stage of growth based upon their peak growth spurt (Table 4) the highest percentage of male runners and controls fell in their peak growth spurt. The fact that there were no MC in the pre growth spurt may explain why the height of MC was greater than MR (Table 3). The highest percentage of FR also fell in their peak growth spurt, but the highest percentage of FC fell 61 62 in their post growth category (Table 4). This could explain why the height of the controls was greater than FR (Table 3). In Table 4, although female subjects in the pre and peak groups had similar mean increases in height, the group in the pre growth category was not placed in the category of those who were in their peak growth because their pattern of growth had been steady, at a constant rate, and they have not yet undergone a sharp acceleration in growth, as indicated from their plotted heights (from the past 3 phases and the phase following Phase III) on the NCHS growth charts (111). The spurt of growth indicates that the adolescent has reached puberty. The Tanner ratings were checked for these two subjects, and may also indicate that the 2 subjects were pre-puberty (1 subject had a Tanner rating of I and the other subject had a rating of II), reenforcing the classification of these subjects in the pre growth spurt category. There was one PC in the pre growth spurt category with an increase in height of 8.3 cm. from Phase II to Phase III (dcm/dt). This individual had been growing at a steady, but rapid rate; her growth Spurt did not occur until the following year (as indicated by her height measurement taken in Phase IV, Fall, 1985). Her Tanner rating (Tanner rating was I) also indicated that she had not yet reached puberty. When subjects were classified according to growth categories, based on the shape of their growth curve, it appeared that runners and controls reached their growth spurt at the same age for both genders (Table 5). In any case, runners did not have a delayed growth spurt, because there was a trend for them to be younger when they reached their growth spurt than their counterparts of the same gender. Both 63 male and female controls tended to be taller than runners during the peak growth period but height equalized in the post growth category. Controls were significantly heavier than runners of the same gender in the peak growth period. In the post growth period there was a similar trend but it was not significant. In Phase III, the mean relative weight for age of MC was significantly greater (p<0.01) than that of the MR (Table 6). MR had a mean relative weight 8% below the 50th percentile of weight for age and MC had a mean value 12% above the 50th percentile of weight for age. Heights for both groups were at the 50th percentile height for age. FR had a mean relative weight 15% less (p3. 3).-05 92.2. .855 a=(p<0.01) 115 Figure 9. Mean daily protein intake, for MR, MC, FR, and F0 for Phases II and III. Phase II: MR, N=21 MC, N=10 FR, N=13 FC, N=9 Phase III: MR, N=20 MC, N=11 FR, N311 FC, N=10 Figure 10. Mean daily protein intake, for subjects categorized into pre-, peak-, and post- growth spurts for MR, MC, FR, and PC for Phase III. MR Pre, N=5 MC Pre, N=O FR Pre, N=2 FC Pre, N=1 Peak, N=13 Peak, N=9 Peak, N=6 Peak, N=4 Post, N=2 Post, N=2 Post, N=3 Post, N=5 116 a W////%////////////// .m m L VAR \ MW...» 8. “has \\ m... . Veg/é m m 8 m. 8 “W\\\\\\\\\\\ \ a m . § . .m L V////V/////////.%////////////m a Z? W MIMI/l ./ A .M qqdufi qd—qfid- mmmmuuaauunaa. nmmmuaaamnaeu. BBBBB 2 1.1.1:“ .3: 5 2.22... . . A3: 5 2.22... 117 Figure 11. Mean daily dietary iron intake, for MR, MC, FR, and PC for Phases II and III. Phase II: MR, N=21 MC, N=10 FR, N=13 FC, N=9 Phase III: MR, N=20 MC, N311 FR, N=11 FC, N-IO Figure 12. Mean daily dietary iron intake, for subjects categorized into pre-, peak-, and post- growth spurt for MR, MC, FR, and PC for Phase III. MR Pre, N=5 MC Pre, N=0 FR Pre, N=2 FC Pre, N=1 Peak, N=13 Peak, N=9 Peak, N=6 Peak, N=4 Post, N=2 Post, N=2 Post, N=3 Post, N=5 118 .\\\\\\\\\\\\\\ ammnuuuuuw. .3563»! a=(p<0.10)° b=(p<0.10) 1221-! tSSIIc m 1. M "3 -rn W‘“\+~\k‘ \ WW //////////// R\\\\\\\\\\\. x// / 7V//////////////// 7”””’ - q ‘ ‘ M ‘ qua-Ju-qqd- mnnuuwuumo 93.. 5 .8... >563 ///////.. 119 Figure 13. Subjects were divided into 8 categories based upon their mean daily iron intakes in mg. This figure illustrates the percentage of subjects who fell within each category in Phase II. Phase II: MR, N=21 MC, N=10 FR, N=13 FC, N=9 Figure 14. Subjects were divided into 8 categories based upon their mean daily iron intakes in mg. This figure illustrates the the percentage of subjects who fell within each category in Phase III. Phase III: MR, N=20 MC, N=11 FR, N=11 FC, N=10 120 18 mg. RDA '- 7’]. MALE RUNNERS 7::_, sw mmm r/, RNN 1mm mm... A .\ EEK '/. llllllll mil/Viz \ _ ._____:::_a g, / z_==_____==_=_==__§ __ z A: 2 / .\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ wianumumwwso 25$ 33 «50:0 mun. gmsmaw 12.1-15 15.1-18 18.1-21 21.1-24 24.1-27 >27 9.1-12 5-9 s m S R O a a. m m m m w w W m R C F. C E M . E i m. m m m 0.. M. M M m H w-EN .::____fi m . /, W. I m. \\ \\ m m 8 IE 1 % WH III. IIIIIIIIIIIIIIIIIIIIII R P m H R sagas-.__... R m E E u a , . _ i 2 _: __ __:__:::_2,23: 7 /. §\\\\\\\\ \\\, 7 7 . . _ ,J,_,m::_:1_;_f / Figure 13. wwawumumwwso 3.. .505 mm... meamam 5-9 24.1-27 >27 21.1-24 18.1-21 PHASE III 15.1-18 DIETARY IRON INTAKE / DAY (mg.) 12.1-15 9.1-12 Flam 14». 121 Figure 15. Mean daily total protein, animal protein, and plant protein intakes for MR, MC, FR, and FC, for Phase III. Phase III: MR, N=20 MC, N=11 FR, N=11 FC, N=10 122 7’. W//////////////////// §.. §% 1.“ .D %kl ” qqqqqqqqqq PLANT PROT a=(p<0.01) b=(p 75; (%) 8 (EF) = 0; (%) 2111 mg; Storage Level (EF) < 75; (x) 4 + 14.3logn(EF+IOO)/IOO. (EF) > 75; (%) 12 (EF) 8 O; (%) 4 O m; Storage Level (EF) < 75; (%) 5 + 26.8 logn(EF+100)/100 (EF) > 75; (%) 20 (EF) 2 0; (%) 5 3 An illustration Of the estimated percentages associated with each category of iron status, as calculated by this model, Of the two extremes and the average level of availability of iron in a meal are displayed in the table on the following page. 165 APPENDIX B MONSEN'S MODEL (18); A METHOD TO CALCULATE PERCENT or ABSORBABLE IRON IN A MEAL Table 81. FACTORS FOR ESTIMATING PERCENT ABSORPTION OF DIETARY IRON AT EACH IRON STORAGE LEVEL OF 0 MG., 250 MG., AND 500 MG. IRON STORAGE LEVELS 0 mg. 250 mg. SOOamg. I. Heme Iron 35% 28% 23% II. Nonheme Iron A. Lowest Availability Meal 5% 4% 3% MFPb + Ascorbic Acid = o B. Medium Availability MEal 10% 7% 5% MFP + Ascorbic Acid = 1 - 75 C. 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