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The effects of prelatent and latent iron deficiency on physical work capacity Newhouse, Ian Joseph 1987

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THE EFFECTS OF PRELATENT AND LATENT IRON DEFICIENCY ON PHYSICAL WORK CAPACITY By IAN JOSEPH NEWHOUSE B.P.E., The University of Alberta, 1980 M.Sc, The University of Alberta, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES INTERDISCIPLINARY STUDIES (PHYSICAL EDUCATION/MEDICINE/ZOOLOGY) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October 1987 © I a n Joseph Newhouse, 1987  In  presenting  degree  at the  this  thesis in partial fulfilment of  of  department  this thesis for scholarly or  requirements  for  by  his  or  her  I further agree that permission for  purposes  advanced  representatives.  permission.  Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  extensive  may be granted by the head of It  is  understood  that  publication of this thesis for financial gain shall not be allowed without  DE-6(3/81)  an  University of British Columbia, I agree that the Library shall make it  freely available for reference and study. copying  the  copying  my or  my written  ABSTRACT  In order to  examine the effects of prelatent/latent iron deficiency on  physical work capacity and selected muscle enzyme activities, forty female subjects were studied before and after supplementation  eight weeks of  with either oral iron or a matching placebo.  Initially, female volunteers engaged in regular endurance running were screened for  iron deficiency by blood analysis (serum ferritin  and hemoglobin).  Forty non-anemic subjects with deficient iron  stores underwent physiological and anthropometric tests to obtain a comprehensive profile.  The specific physical work capacity tests  were alactic and lactacid power on the Wingate cycle ergometer test, lactacid capacity on the anaerobic speed test, anaerobic (ventilatory) threshold using gas exchange variables, V 0 treadmill velocity during the  2  max. and the max.  v"0 max. test. Muscle biopsy samples  pre-, and post- treatment were  2  assayed for citrate synthase and  alpha-glycerophosphate dehydrogenase activity.  Treatment was oral  iron supplementation (320 mg ferrous sulfate = 100 mg elemental iron taken as SLOW-Fe® twice a day) or a matching placebo. The subjects were randomly assigned to one of the treatment groups and a double-blind method of administration of the supplements was used.  ii  It was hypothesized that work capacity would be enhanced following oral iron supplementation, possibly due to the repletion of iron containing oxidative enzymes important in energy production. could  not  strongly  support  this  hypothesis  with  the  Results  difference  between the two groups on the work capacity and enzyme activity variables being statistically nonsignificant. rose  from  a  mean  Serum ferritin  of  12.4+4.5 to  37.7+19.7 n g m l "  and  12.2±4.3 to  17.2±8.9 for the  values for the  1  experimental  group  (p=0.0025).  Hemoglobin levels remained fairly constant for both  treatment groups;  13.4±0.6 to 13.5±0.5 g d l "  13.0±0.6 to 13.1+0.5 (control); (p=0.6). capacity  variables,  experimental  (experimental),  and  Pre to post values on the work  vs  Alactic power, 8.8 to 8.4 watts-kg"  1  controls;  control  respectively  were:  body wt. vs 8.4 to 8.2; lactacid  1  capacity, 6.9 to 6.9 watts-kg" body wt. vs 7.0 to 6.0; anaerobic speed 1  test, 41.3 to 45.1 seconds vs 43.7 to 44.8; anaerobic threshold, 7.4 to 7.5 mileshour"  vs 7.2 to 7.2; V 0 max, 51.3 to 52.7 ml-kg" min"  1  vs  50.6 to 50.6; max velocity during V 0 max, 9.8 to 9.8 mileshour"  1  vs  1  1  2  2  9.6 to 9.5.  Except for alactic power, the change in work capacity  favored the iron treated group. be warranted.  Prelatent/latent  Noting this trend, further study may iron deficiency  appeared  depress the activities of the two enzymes measured. alpha-glycerophosphate dehydrogenase  not to  Cytoplasmic  activity rose from 0.066 to  0.085 units for the experimental group (p=.58) vs .058 to .066 for the control group and citrate synthase activity changed from 0.047 to 0.048 (experimental) vs 0.039 to 0.042 (control). iii  It can be concluded that eight weeks of iron supplementation to prelatent/latent  iron deficient, physically  active females does  not  significantly enhance work capacity nor the activity of 2 oxidative muscle  enzymes  alpha-glycerophosphate  (citrate  synthase  dehydrogenase).  and  cytoplasmic  Within the limitations of  this study the presence of a serum ferritin below 20 ng-ml"  1  pose a significant handicap to anaerobic or aerobic capacity.  iv  does not  TABLE OF CONTENTS  Page  Abstract  ii  List of Tables  vii  List of Figures  viii  Acknowledgements  ix  Introduction  1  Methods  5  Results  19  Discussion  35  References  49  Appendix A. Review of Literature  65  Overview of Iron Metabolism  65  Negative Iron Balance in Athletes  69  Iron Deficiency With Anemia  81  Iron Deficiency Without Anemia  85  B. Definitions  99  C. Limitations  104  D. Delimitations  106  v  Page E. Subject Consent Form  107  F. Muscle Biopsy Consent Form  110  G. Dietary Analysis  111  H. Schedules of Examinations and Tests  11 6  I. Iron Status Raw Data  117  J. Work Capacity Raw Data  11 8  K. Enzyme Activity Raw Data  11 9  L. Descriptive Statistics of Placebo Treated Subjects who became Anemic  1 20  vi  LIST OF TABLES  Page  Table I.  Descriptive Statistics of Iron Status  22  Table II.  Descriptive Statistics of Work Capacity  24  Table III.  MANOVA on Iron Status  27  Table IV.  MANOVA on Work Capacity  28  Table V.  Comparison between the Latent Iron Deficient Females within the Iron Treated Group and the Placebo Group on the Anaerobic Speed Test  vii  30  List of Figures  Figure 1.  Page  Distribution of Serum Ferritin Levels among the Females initially Screened for Inclusion in the Study  Figure 2.  19  Distribution of Hemoglobin Levels among the Females Initially Screened for Inclusion in  Figure 3.  the Study  20  Iron vs Placebo Summary  26  viii  Acknowledgements  The efforts, advice and expertise of a large number of people have made  this  thesis  possible.  I would  especially  like  to thank  CIBA-Geigy pharmaceuticals who funded this research and helped formulate the proposal. was considerable. time,  exhaustive  The commitment required from the  subjects  I am deeply indebted to these volunteers for their physical  efforts  donated to the cause of the study.  and muscle biopsy  specimens  The guidance provided by Dr. Doug  Clement and my research supervisor, Dr. Don McKenzie was invaluable, as was the input of each of my supervisory committee members in their own areas of expertise.  I am also grateful to the technical  assistance provided by Dr. Gord Matheson, Dr. Tom Momson, Lynne Sawchuk, Lois Culling and Dusan Benicky.  ix  1  INTRODUCTION  Iron deficiency is the most common form of nutritional deficiency in many populations (Siimes et a l . , 1980). prevalence and severity  Although  it reaches its greatest  in developing countries, it is also frequently  encountered in affluent societies.  According to the Nutrition Canada  Survey of 1973, 76% of Canadian women had iron intakes below the recommendation for the general population. that regular exercisers  Recent literature suggests  may be at increased risk of iron deficiency  (Clement and Asmundson, 1982; Dickson et al., 1982; Ehn et al., 1980; Wishnitzer et al., 1983).  Based on serum ferritin levels, which are  believed to accurately reflect the size of the body iron stores (Jacobs, 1977), it was observed  that 82% of female, elite Canadian distance  runners were iron deficient (Clement and Asmundson, 1982). report  involving  long  distance  runners  found  that  Another  despite  normal  hemoglobin and serum iron values, the bone marrow showed either an absence or only traces of iron (Ehn et a l . , 1980). investigators  have confirmed this surprisingly  Several  other  high incidence of iron  deficiency in active persons, particularly endurance runners (Dickson et al., 1982; Nickerson and Tripp, 1983; Wishnitzer et al., 1983).  As women are already  at increased risk due to the superimposed  requirements related to menstruation, the possibility of an increased iron demand associated with exercise is of particular concern to those engaged in regular physical activity.  As a result, a variety of supplementation  2 regimens and monitoring schedules are recommended to ensure adequate iron status (Bothwell et al., 1979)  These intervention programmes, aimed  at reducing the prevalence of iron deficiency, are predicated on the assumption that nutritional iron deficiency does indeed lead to significant disability.  In the case of iron deficiency with anemia, there is no doubt that the condition is undesirable.  Several studies in rats and in humans (Anderson  and Barkve, 1970; Edgerton et al., 1972; Viteri and Torun, 1974) have shown a significant correlation between the hemoglobin concentration and work capacity, with some results indicating that even minor decrements affect performance.  This decreased physical work capacity in anemic  subjects was presumably due to the reduction in oxygen delivery to the musculature (Anderson and Barkve, 1970).  However, more recently, the effects of iron deficiency without anemia on physical work capacity have been examined and this condition has been shown to reduce exercise tolerance or blood lactate levels at maximal exercise (Finch et al., 1976; Finch et al., 1979; Schoene et al., 1983). Finch et al's. studies used exchange transfusions to bring the hemoglobin levels  of anemic rats  back to  normal  levels.  This  iron  deficient  non-anemic state would not replicate the prelatent/latent iron deficiency observed in many athletes for in the rat studies this state was achieved artificially and the prior state of iron deficiency was severe.  The studies  did show though that despite normal hemoglobin levels, iron deficient rats had a marked impairment of running ability as compared to controls.  This  3 defect in physical performance was attributed to reduced concentrations of an  iron containing  enzyme  (mitochondrial  alpha-glycerophosphate  dehydrogenase: a-GPDH) important for energy metabolism (Finch et al., 1976). system  Mitochondrial a-GPDH is a component of the electron transport and  is  thought  of  the  cytosol  and  Furthermore, it appears that when the activity of  this  alpha-glycerophosphate mitochondrion. enzyme  is  decreased,  to  be  shuttle  there  is  the  for  an  limiting  NADH  enzyme  between  accumulation  of lactic acid in the  muscles and this contributes to the muscle dysfunction (Finch et al., 1979).  Schoene et al., (1983) studied the effect of two weeks of iron therapy on exercise performance and exercise induced lactate production in trained, mildly  iron  unchanged  deficient  women  after therapy  athletes.  although  exercise decreased significantly.  Exercise  blood  performance  lactate levels  was  at maximum  The short period of iron repletion may  explain the non-significant change in exercise performance.  Valberg  (1980) noted that oral iron treatment needs to be continued for two months to replenish iron stores after the hemoglobin concentration has reached normal values.  The need for investigation involving non-anemic,  iron-deficient, human subjects is a pressing one since the results have far-reaching implications in relation to the well-being and productivity of large  population  reproductive years.  groups,  particularly  exercising  females  in  their  4 Thus the purpose of this investigation was to determine if iron deficiency without anemia limits physical work capacity.  The specific aim was to  assess the anaerobic/aerobic work capacities of prelatent/latent  iron  deficient, physically active females before and after eight weeks of oral iron supplementation.  Assuming that changes in exercise tolerance may be  due to repletion of iron-containing oxidative enzymes important in energy production (Finch et al., 1976, 1980), the activity of cytoplasmic alphaglycerophosphate  dehydrogenase was measured before and after the  treatment period to give an estimate of the maximum possible flux through the NADH-glycerophosphate shuttle system.  This shuttle system  serves two purposes; 1. regeneration of nicotinamide adenine dinucleotide ( N A D ) necessary for the continued operation of the glycolytic cycle and 2. +  generation  of adenosine 5'-triphosphate  a-glycerophosphate  (ATP)  by oxidative phosphorylation  via the  oxidation  of  (Finch et al., 1976).  Citrate synthase, which was also assayed to assess change in activity, is a regulatory enzyme which catalyzes the first reaction of the Krebs cycle, the  condensation  (Lehninger, 1982).  of  acetly-CoA  with  oxaloacetate  to  form  citrate  It was hypothesized that physical work capacity would  be enhanced following eight weeks of oral iron supplementation, possibly due to the repletion of iron dependant enzymes important in aerobic metabolism.  5  METHODOLOGY  1.  Subjects  Healthy,  physically  active  female  runners  from  the greater  Vancouver area formed the population from which a sample was drawn.  Forty volunteers between the ages of 18 and 40 years were  selected after screening  155 females for prelatent/latent  deficiency (i.e., serum ferritin below 20 ng-ml" level of 12 g d l " or greater).  1  iron  and hemoglobin  It should be noted that no condition  1  except iron deficiency has been reported to produce a low serum ferritin concentration (Valberg,  1980).  The subjects signed informed consent forms (See Appendices E and F) and all procedures were approved by the University of British Columbia Ethics Committee.  Criteria for exclusion from the study were occult blood loss in the stool  as determined  from  serial  fecal  specimen  analysis  (Hemocult, Smith-Kline Diagnostics, Inc.) or blood loss in urine as determined by color changes  on a dip and read chemstrip  (Chemstrip, Boehringer Mannheim Canada Ltd.). exclusion  were:  ingestion  Other reasons for  of acetylsalicylic acid (aspirin) or  medication which may cause acute or chronic blood loss from the gastro-intestinal  tract  (such  as  other  non-steroidal  6 anti-inflamatory agents, steroids or solid potassium supplements) one week prior to the pre-test or during the treatment period; pregnancy, blood donation or loss of more than 500 ml of blood through injury within one month of the pre-test or during the treatment period;  dietary intakes of vitamin C (ascorbic acid) of  less than 60 m g d a y " analysis;  1  as determined from three day dietary  taking of a birth control pill coincidental with a low  serum folate (below 3.2 nmol'L" ); and finally a fever within 2 1  weeks of the hematological pre-test.  Data from protocol deviators was excluded from the  analysis.  Protocol deviations included: failure to take at least 7 5 % of the medication (as determined by pill count); failure to return for post-testing within five days of scheduled date; and development of a fever (oral temperature greater than 38.5° C) within two weeks of the post-test.  Subjects who failed to complete the full eight weeks of the study for reasons of toxicity, lack of compliance, lack of cooperation or reasons other than treatment failure were considered dropouts. Seven subjects dropped out with the reasons being injury (3), lack of compliance (2), and relocation (2). until the completed.  Patient accession continued  requisite number of evaluable study Continuous  subjects  were  enrollment ensured that 40 subjects  completed the experimental procedures.  7  2. Test  Items or  Subjects  Instruments  underwent  hematological,  anthropometric  physiological tests to obtain a comprehensive profile.  and  Three work  capacity tests were chosen to assess the range of highly anaerobic to highly aerobic function.  a)  The test items included:  Hematological data:  Hemoglobins  were  performed  on the ELT-8  multiparameter  instrument (Ortho Diagnostic systems, Inc., Raritan, New Jersey) by  a  colorimetric  cyanmethemoglobin  method.  Hemoglobin  by the addition  is  converted  of a reagent  potassium ferricyanide and sodium cyanide (cyanac).  to  containing The density  of the color produced is directly proportional to the amount of hemoglobin present (Sonnenwirth and Jarett, 1980). uses  a  modified  hemoglobin.  cyanmethemoglobin  method  The ELT-8 to  measure  Light is transmitted through a cuvette containing a  sample of blood diluted with cyanac reagent.  The amount of light  that passes through the sample is measured by a light sensor. The output of the sensor when measuring the specimen is compared to that of the reagent blank. hemoglobin in the sample.  The difference represents the amount of  8  Serum  ferritin  concentration  was  determined  by  the  2 site  Quantimune® Ferritin immunoradiometric assay (IRMA) (Bio-Rad Laboratories, Mississauga, Ontario).  IRMA is based on principles  described by Bothwell et al. (1979) and Addison and Hales (1971). After  raising antisera in rabbits against  ferritin, an antibody  human  to ferritin is isolated by  recrystallized  mixing immune  serum with ferritin that has previously been rendered insoluble by allowing it to react with diazocellulose.  After incubation, the  specific IgG molecules extracted in this way are labelled with while still coupled to the insolubile ferritin.  The  1 2 5  l  1 2 5  l  labelled  antibodies are then eluted by acidification for use in the routine assay.  In the routine assay ferritin standards are incubated with  the unknown serum and immunoadsorbent (ie.  125  l-labelled antiferritin IgG.  Ferritin  binds and insolubilizes any reacted  with  centrifugation.  ferritin  and  A ferritin  Immunobeads®) is added which  125  l - l a b e l l e d antibody that has not  enables  it  Supernatant radioactivity  to  be  removed  by  is proportional to the  amount of ferritin in the sample being tested.  Based on repeated  measurements of quality controlled sera in 20 consecutive assays, the between assay variation is about 8 to 10 % (Miles et al., 1974).  Total iron (serum iron) measurement is based on the principle that in an acidic medium, transferrin bound iron dissociates into free ferrous and ferric ions.  Ascorbic acid is then used to reduce the  ferric iron to the ferrous state.  Ferrozine®  (3-(-pyridyl)-5,6-bis  9 (4-phenylsulfonic  acid)-1,2,4 triazine, disodium salt) reacts with  ferrous iron to form a magenta colored complex which absorbs at 560 nm.  The absorbance is directly proportional to the amount- of  iron in the serum (Sonnenwirth and Jarett, 1980)  Reagents for  both total iron and unsaturated iron binding capacity (UIBC) were obtained from Canada.  Diagnostic Chemicals Ltd., Charlottetown, P.E.I.,  Performance characteristics as reported  by the kit  manufacturer list the coefficient of variation as between 1.8 and 0.6% for total iron and between 1.4 and 3.7% for UIBC.  Unsaturated iron binding capacity (UIBC) is determined as follows: A known ferrous iron standard containing 70 u.mol-L" (391 jigdl" ) 1  1  incubated with the serum at a pH of 7.9 saturates the available binding sites on serum transferrin.  The unbound excess iron is  then complexed with ferrozine and the UIBC is determined. UIBC is equal to the total iron added less the excess iron.  The Total  iron binding capacity (TIBC) is equal to serum iron plus UIBC.  % Transferrin saturation is arrived at mathematically by dividing total iron by TIBC.  All hematological work was done at the University of British Columbia Health Sciences Centre Hospital.  Subjects reported to  the lab in the morning having avoided physical activity in the preceding 24 hours.  10 b)  Anthropometric data:  Percent  body  fat  (densitometry),  height  (Holtain  unclothed weight (Detecto Scales) were recorded.  Ltd),  and  This data was  recorded for descriptive rather than statistical purposes.  c)  Physiological tests:  The  research facility used for physiologic (and anthropometric)  testing was the Buchanan Fitness Laboratory University of British Columbia campus.  located on the  The physiological tests  were spread over 2 visits with the Wingate and the anaerobic speed test performed on the first visit ( „ between  the  two  tests)  and  the  45 minute  recovery  incremental treadmill test  performed on the second visit.  The Wingate anaerobic power test measured alactic anaerobic capacity and lactacid capacity on the cycle ergometer.  A Monark  bicycle ergometer equipped with toe clips and an electrically triggered  counter  were  used  in this  test.  Subjects  were  instructed to pedal as fast as possible for the 30 second test duration.  Within 2 to 3 seconds of fast pedalling, the resistance  was adjusted to 75 g-kg"  1  and counter were activated.  body weight.  At that instant the timer  Vocal encouragement was given.  number of pedal revolutions was recorded every 5 seconds.  The  11 Alactic power corresponded to the maximal power averaged over a 5  second  period  (Watts-kg"  corresponded  to the average  (Watts-kg"  body  1  body  1  wt.).  Lactacid  power  power output over 30 seconds  wt.),(Bouchard  et al., 1982).  Test-retest  reliability is reported to be 0.90 to 0.93 for a 1 or 2 week interval (Bouchard et al., 1982).  The Anaerobic speed test on  the treadmill measured lactacid  capacity and was usually of a longer duration than the Wingate test (ie. 30-60 seconds). As most subjects were runners, this test was more sport specific.  A Quinton 24-72 treadmill set at 7  miles-hour" and 20% grade was used. 1  run  as  long  exhaustion.  The  as  possible with  Subjects were instructed to  results  recorded  as time to  Reliability is 0,76 to 0.91 (Bouchard et al., 1982)  progressive  workload treadmill test  anaerobic/ventilatory  assessed both  threshold using expired gas analysis and  vT^max as the workload increased to the point of exhaustion. final treadmill velocity  achieved was  The  also noted as a work  capacity variable. Subjects warmed up on the treadmill least 5 minutes.  the  for at  The initial treadmill speed was 5.0 miles-hour"  1  and increased by 0.5 miles-hour" every minute; the treadmill grade 1  remained at 0% for the duration. direct  chest  lead  EKG  (Burdick  Heart rate was monitored by EK/5/A  electrocardiograph).  Expired gases were sampled and analyzed by a Beckman Metabolic Measurement Cart interfaced to a Hewlett Packard 3052 A Data  12 Aquisition System.  Two  major  criteria were  used  to  determine  the  anaerobic  threshold: 1) a non-linear increase in excess C 0 (ie. V C 0 2  V0 )) 2  2  and 2) a non-linear increase in ventilation (ie.  - (RQ X Ve/V0 ). 2  Ventilatory and gas exchange variables were tabulated every 15 seconds for the duration of the treadmill test allowing for plots of these variables against the speed of the treadmill.  Results were  expressed as the velocity of the treadmill (mph) at the anaerobic threshold. Two independent evaluators determined the anaerobic threshold. of opinion.  A third evaluator arbitrated irreconcilible differences A test-retest correlation for this variable was found  to be 0.91 (Davis et al., 1979).  V0 max 2  was determined by the mean of the four  consecutive 15 second values.  highest  Thoden et al. (1982) report a  test-retest correlation of 0.95. d)  Muscle Biopsies  This was  an optional procedure  volunteered and Information  for all subjects;  17 of 40  utilized a separate consent form (see Appendix F).  gained from this aided in understanding  possible  biochemical effects of prelatent and latent iron deficiency.  13 Two  biopsy  samples were  post-treatment).  The  portion of the right cm above the knee.  thus  taken  biopsies were  (one  pre-  and  one  taken from the lateral  quadriceps muscle (vastus lateralis) about 20 First a small area  local anesthetic (2% xylocaine).  was anesthetized with  An incision was made large  enough to allow the biopsy needle to be put into the muscle. Approximately 50 mg of muscle was excised with the needle and was immediately frozen in liquid nitrogen and stored at -80°C for further analysis.  The biopsy excision was conducted at the University of British Columbia Health Science Centre Hospital and the assays were performed in a laboratory of the Zoology  department at the  University of British Columbia.  Assays  were  performed  for  citrate  synthase  activity  and  alpha-glycerophosphate dehydrogenase (a-GPDH) activity. After  thawing,  samples  were  homogenized  with  Polytron  homogenizer (2 bursts of 10 seconds) in 10 volumes imidazole buffer (50 mM Imidazole, adjusted with HCI to pH 7.8 at room temperature).  Homogenates were centrifuged briefly (60 seconds  at 13,000 x g) and aliquots of the supernatants were used for the enzyme assays without further dilution.  14 Citrate  synthase  (E.C.,  citrate  oxaloacetate-lyase  [CoA-acetylating]) was assayed at 412 nm, utilizing the reduction of  DTNB  (5,5'-dithiobis- (2-nitrobenzoic  liberated as the indicator reaction.  acid)  by the  HSCoA  Final concentrations of the  reactants and cofactors in the reaction medium were: 50 mM Tris-HCl (pH 8.1), 0.1 mM DTNB, 0.25 mM acetyl-Coenzyme A, 0.5 mM oxaloacetate (omitted for control).  The extinction coefficient  of DTNB at the chosen pH and wavelength is 13.6 cm -|iMol" . 2  Alpha-glycerophosphate dehydrogenase (E.C.  1, sn-glycerol  3-phosphate: NAD 2-oxidoreductase) was determined at 340 nm, following  the  oxidation  of  dihydroxyacetonephosphate  NADH  upon  (DHAP).  The  the  addition  reaction  of  medium  consisted of: 50 mM Imidazole-HCI (pH 7.8), 0.15 mM NADH and the dimonocyclohexylamine salt of DHAP at a final concentration of 1 mM (omitted for control).  The extinction coefficient of NADH at  the chosen wavelength is 6.22 cm -u.Mol" . 2  Final reaction volumes were followed for 1 - 5 min.  1  1.0 ml and reaction rates were  It was ascertained that rates were linear  with time and with volume of homogenate used.  Determinations  were done using a Unicam SP6-500 recording spectrophotometer fitted with a thermostatted cell holder and a Soltec single channel recorder.  Cells were maintained at 25°C; all reactions were done  in duplicates.  One unit is defined as the amount of substratye  converted to product per minute at 25°C in the presence of  15 saturating concentrations of substrates (Vmax).  All steps in the preparation of extracts were performed at 0 - 4°C. Enzyme  activities  were  measured  within  2  hours  of  homogenization.  Protein  concentration  in  the  supernatants  of  the  muscle  homogenates was determined following the procedure outlined by Bradford (1976).  The technique employs Coomassie Blue as the  color reagent and 5 volume of 1 ml.  JLLI  of homogenate were used in a final assay  Defatted bovine serum albumin was used as a  standard.  After initial testing, individuals conforming to entry criteria were randomly assigned to one of two treatment groups.  The two  treatment groups were: a) Experimental:  The experimental treatment consisted of  oral iron supplementation (320 mg ferrous sulfate = 100 mg elemental iron) taken as SLOW-Fe® 1 tablet twice a day, as tolerated.  This tablet appeared different from SLOW-Fe® that is  available commercially. b) Control:  The control group took a placebo which appeared  identical to the active agent and was taken 1 tablet twice a day as  16 tolerated.  Ingredients:  Cornstarch, lactose, gelatin, talc, stearic  acid, magnesium stearate, green #3 dye, silicon dioxide, edible shelac, hydroxypropylmethyl cellulose, PEG 4000.  A double blind method of administration and random assignment to treatment groups was used.  This involved computer generated  randomization with each enrolled participant assigned to the next consecutive number.  Treatment continued for eight weeks with a  follow-up visit at four weeks and retesting upon completion of treatment. training  the  treatment  period  (type, duration,  intensity)  and  (estimated  During  from  number  subjects  menstrual  of tampons/pads  monitored blood  used each  loss  cycle).  Subjects were instructed to make no changes in their training programme or their diet during the study. analysis  prior  to  both  the  pre-test  and  A three day dietary post-test  aided  in  monitoring diet. (See Appendix G)  The post-testing procedures were identical to the initial testing. See Appendix H for schedules of examinations and tests.  3. Data  Analysis  The experimental design is a 2 x 2 (treatment x time) factorial experiment with repeated measures on the second factor as shown on the following page.  17  EXPERIMENTAL  S 1 j  n=20  n=20  n=20  n=20  PRE-TEST  POST-TEST (8 WEEKS)  S 20 S 21 CONTROL  I S 40  TIME  The independent variable was oral iron supplementation. There were 11 dependent variables with two domains (iron storage and work capacity) and thus two multivariate analyses of variance were used.  This provided experiment-wise control over the type  one error rate. chosen.  A significance probability level of 0.05 was  The dependent variables were:  IRON STATUS: a)  Serum ferritin levels  b)  Hemoglobin levels  c)  Total iron  d)  Unsaturated iron binding capacity (UIBC)  e)  % Transferrin saturation  18 WORK CAPACITY: f)  Alactic capacity on Wingate cycle ergometer test  g)  Lactacid capacity on Wingate cycle ergometer test  h)  Lactacid capacity on anaerobic speed test  i)  Anaerobic threshold on treadmill  j) V 0 k)  2  max on treadmill  Max treadmill velocity during V 0 m a x test. 2  A cannonical correlation was used to obtain a measure of the linear association between improvement scores on the domains of iron storage (serum ferritin and hemoglobin) and work capacity (variables f-k above).  The loadings associated with each variable  in the cannonical variate indicate the relative contribution that each  variable within  the set makes toward  the relationship  between the two sets (Schutz et al., 1983).  The  statistical programs  Software, Inc. , Los Angeles.  used  were  from  BMDP Statistical  19  RESULTS  One hundred and fifty-five physically active women (age 18-40) underwent blood testing to select a sample population who fulfilled the criteria of prelatent/latent iron deficiency. (Figures 1 and 2)  The slightly smaller  number of subjects shown in these figures is due to subjects who obtained a serum ferritin determination but no hemoglobin value or vice versa.  40 Number Of Subjects (n= 152)  30 25  II I I I I I I I  llllll I  50 | 70  I  40 60 80 Mean (29.9)  90 | 1 1 0 | 130| 150| 170| 190 100  120  140  160  180  Serum Ferritin (ngml ) -1  FIGURE 1: Distribution of serum ferritin levels among the females initially screened for inclusion in the study.  20 The inclusion criteria for serum ferritin levels was 20 ngml"  1  or less.  Sixty-one of the subjects (40%) fit this classification while 94 females (62%) had serum ferritin values below 30 n g m l " . 1  Two subjects were  above the lab's normal range of 10 to 160 ngml" while 20 subjects were 1  below this range.  FIGURE 2: Distribution of hemoglobin levels among the females initially screened for inclusion in the study.  21 Hemoglobin values among the females were centered around the mean of 13.0 g d l " . 1  Thirteen females (8.5%) could be classified as anemic (Hb  below 12.0 gdl" ). Twelve of these women had low iron stores as reflected 1  by serum ferritin values below 30 ngml" . 1  On the basis of hemoglobin and ferritin measurements, 47 females qualified to continue with the study.  Forty of the selected females completed the eight week study, 19 of whom received iron supplements and 21 of whom received matching placebos. Pre and post measurements of iron status (Table I) and work capacity (Table II) were taken.  22  TABLE I Variate Serum ferritin (ng-mr )  Group  Count  Pre-Test  Post-Test  Iron  19 18  12.4±4.5 12.2±4.3  37.7119.7 17.218.9  19 18  13.4±0.6 13.0±0.6  13.510.5 13.110.5  13 14  79.8±48.7 84.1143.0  98.5140.1 70.5145.7  13 14  251.8±81.0 252.4161.5  195.7156.1 231.9143.9  13 14  25.1112.8 25.6112.9  34.1114.4 21.4113.0  1  Placebo Hemoglobin (gdl" ) 1  Iron Total (ug-oT )  Iron Placebo  1  Iron UIBC (ugdr ) 1  % Saturation (%)  Placebo Iron  Placebo Table I: Descriptive statistics of iron status, Iron mean and l S.D.)placebo group (count, Placebo  iron group  vs  23  Serum ferritin values rose from a mean of 12.4 to 37.7 for the iron supplemented group, but also noteworthy is the ±19.7 standard deviation during the post-test. treatment groups. were  discarded)  Hemoglobin values remained constant for both  Due to missing pre-test values, (the blood samples the  count  for the  iron  profile  measurements  was  decreased to 13 (iron) and 14 (placebo).  Alactic power dropped slightly for both treatment groups while only the placebo group showed a performance decrement on the lactacid variable.  The  capacity  iron treated group improved their performance on the  anaerobic speed test by almost 4 seconds compared to an improvement of just over  1 second  for the controls.  The  incremental speed  test  measurements of anaerobic threshold and max. treadmill velocity remained almost constant for both treatment groups.  On the V 0  2  max variable a  modest improvement was posted (51.3 to 52.7 mlkg" min* ) for the iron 1  1  supplemented group, with no mean change noted for the placebo treated group.  Tables of raw data for iron status, physical work capacity and enzyme activity can be found in appendices I, J, and K.  24  TABLE  II  Variate  Group  Count  Pre-Test  Post-Test  Alactic Power (Watts-kg body wt)  Iron Placebo  19 18  8.8±1.0 8.411.0  8.411.1 8.211.4  Lactacid Power (Watts-kg" body wt)  Iron Placebo  19 18  6.910.4 7.010.7  6.910.9 6.010.9  Anaerobic Speed Test (Seconds)  Iron Placebo  19 18  41.3115.2 43.7113.5  45.1116.9 44.8112.9  Anaerobic Threshold (mileshr )  Iron Placebo  19 18  7.411.0 7.2+0.7  7.510.9 7.210.7  19  51.315.7  52.713.8  50.614.9  50.615.5  9.811.0 9.610.7  9.810.8 9.510.7  -1  1  1  V0  2  Iron  Max  (mlkg" min" )  Placebo  Max Treadmill Velocity (during V0 max)  Iron Placebo  1  1  2  18 19 18  (mileshr ) -1  Table II: Descriptive Placebo group.  statistics of work capacity,  Iron group vs.  25 Change scores for each dependent variable were calculated and then standardized (Figure 3 ) by dividing mean improvement by the average standard  deviation  of change.  This  is essentially  an effect  size  measurement which permits valid comparisons between the two groups. The largest positive change was the iron groups' serum ferritin levels. Unsaturated iron binding capacity (UIBC) and % transferrin saturation also showed favorable changes for the iron group. with the work capacity variables.  Smaller changes appeared  For both treatment groups, there was a  performance decrement on the Wingate bike test measurements of alactic and lactacid power.  Anaerobic speed test scores exhibited the largest  improvement with this being the only work capacity measurement on which the control group improved.  Except for alactic power, the change in  work capacity over the treatment period appeared to favor the iron group.  26  2.0  • Iron Group m Placebo Group  1.5  Standard Change 1.0 Score 0.5  i l  =  1 —  —  0.0  -0.5  —  JFer.  Hb.  H  _| — —  1  —  Iron Total UIBC %Sat AlacP LacP AST  AT  702  Max Vel.  Dependent Variables Figure 3: Iron vs Placebo summary. (Fer.-serum ferritin, Hb.-hemoglobin, UIBC=unsaturated iron binding capacity, %Sat.=%transferrin saturation, Alac.P=alactic power, Lac.P= lactacid power, AST-anaerobic speed test, AT-anaerobic threshold, V02=V0 Max, Max Vel.-maximal velocity of treadmill 2  during V0 Max test) 2  Standard change score = mean change score  divided by standard deviation of change.  Two Manova's (Multivariate analysis of variance) were conducted to test the hypothesis that a difference exists between the two treatment groups. In this case the dependent variables are difference scores, pre to post. Table III presents the Manova on change scores with the iron status variables while Table IV presents the Manova on the work capacity variables.  A probability (p) value of 0.05 or less has been chosen to  represent significance.  27  TABLE 111 Effect Overall: (ie. prepost)  Variate Grand Mean -AllChange in Serum Ferritin Change in Hemoglobin Change in Iron Total Change in UIBC Change in % Saturation  Statistic  P-value  T-squared F ratio F ratio F ratio F ratio F ratio  0.0013* 0.0000* 0.9476 0.5962 0.0111* 0.2417  Group: (ie. GXT)  -AllChange Change Change Change Change  T-squared F ratio F ratio F ratio F ratio F ratio  0.0442* 0.0025* 0.6003 0.3482 0.0701 0.1399  in Serum Ferritin in Hemoglobin in Iron Total in UIBC in % Saturation  Table III: MANOVA on Iron Status, highlighted with an asterisk (*).  Significant  P-values are  Serum ferritin levels and UIBC levels were significantly altered for both groups combined (overall effect).  From the group effect, though, the only  significant difference between iron treated and placebo treated groups was the change in serum ferritin levels.  28 TABLE IV Overall Overall: (ie. prepost)  Variate Grand Mean -All Change in Alactic power Change in Lactacid power Change in Anaerobic Spd Test Change in Anaerobic Threshold Change in V 0 Max  Statistic T-Squared F ratio F ratio F ratio F ratio F ratio  0.2409 0.1401 0.4265 0.0239 0.8375 0.3192  Change in Max Velocity  F ratio  0.7068  2  Group: (ie. GxT)  -All Change Change Change Change Change  T-Squared in Alactic power F ratio in Lactacid power F ratio in Anaerobic Spd Test F ratio in Anaerobic Threshold F ratio in V 0 Max F ratio 2  Change in Max Velocity Table IV:  MANOVA on Work Capacity  F ratio  p-value  0.7691 0.6315 0.6291 0.2135 0.5083 0.3116 0.7086  29 There were no significant p-values among the work capacity variables. Although  the Anaerobic  combined)  Speed Test (AST)  scores  improved with an associated p-value  significant T-Squared  (for  both  groups  of 0.024, lack of a  p-value precludes attributing a significant change  in any one variable.  A post hoc ANOVA was added to the analysis to determine if a subgroup of latent iron deficient females within the iron treated group  significantly  improved their work capacity when compared to the placebo group (Table V).  This subgroup consisted of only 5 females with the added criterion  being a % transferrin saturation less than 20% (the normal range is 20 to 55%).  UIBC (321±44u.gd|- ) and total iron (58±11 jj.g d ! )  generally supportive  1  1  values were  of this criterion, although most total iron values  could still be considered normal (40 to 130u,gdl" ). 1  The AST performance  variable was used because the previous MANOVA (Table IV) showed it to be the work capacity measurement closest to significance when comparing treatments.  30  TABLE V Variate Anaerobic Speed Test (seconds)  Group Count Iron 5 Placebo 18  P re-test 34.3±5.6 43.6±13.5  Post-test 38.1 ±8.0 44.8±12.9  P-value 0.38  Table V: Comparison between the latent iron deficient females within the iron treated group and the placebo group on the anaerobic speed test variable.  A significant difference between the groups could not be found, (p-value = 0.38).  This may be due to the small sample and the large standard  deviation, (Table V).  The group means of AST measurements showed an  improvement from 34.3 seconds to 38.1 seconds for the iron treated latent iron deficient females and a more modest improvement from 43.6 seconds to 44.8 seconds for the placebo controls.  31  To further define the nature and strength of the relationship between the domain of iron status and work capacity, two cannonical correlations on change scores were performed.  One evaluation included all iron status  measurements and used a count of 29 while the other evaluation excluded measurements of total iron, UIBC, and % transferrin saturation so as to increase the count to 40. (Note; there were some missing values in these supplementary iron status measurements.) test was not significant.  In either case the Chi-Squared  This indicates that the two sets of variables are  independent.  Of primary interest from the three day dietary records, (Appendix G), was iron intake.  The mean intake of subjects enrolled was 11.8 ± 3.7 mg/day  during the pre-test period and 11.0 ± 3.0 mg/day during the post test period.  Correlations were computed amongst all variables. are presented in table VI.  Selected correlations  Table  VI  Selected Correlations Serum ferritin with total iron(pre) (post) " UIBC (pre) " UIBC (post) " % trans, satn.(pre) (post)  n 30 39 30 39 30 39  r .03 .26 .33 .47 .07 .36  Alactic with lactacid power AST " . . «  40  AST  40  .80 .61 -.67  < .001 < .001 < .001  .02 .23 .09 .25 .15 -.07  > .20 > .20 > .20 < .20 > .20 > .20  "  % body fat  4  0  p (probability) > .20 < .20 <.10 < .02 > .20 <.10  Change scores Serum ferritin with total iron " UIBC " % trans, satn. " AST " AT " V0 Max  30 30 30 40 40 40  Control group only Serum ferritin pre with post Hemoglobin " " Iron total UIBC % Trans. Satn. " " Alactic power " " Lactacid AST AT V0 max.  18 18 13 13 13 18 18 18 18 18  .24 .43 .18 .20 .19 .39 .69 .89 .90 .79  > .20 <.10 > .20 > .20 > .20 < .20 < .01 <.001 <.001 <.001  Max. Workload  18  .81  <.001  2  2  Table VI:  Selected  correlations  33  Seventeen of the forty enrolled subjects volunteered to have muscle biopsy samples taken pre and post treatment from the lateral portion of the quadricep (vastus lateralis).  The extremely small size of two of the  biopsy specimens (ie. <20 mg), prevented analysis of all 17 subjects, while the small size of the samples in general (between 20 to 70 mg) allowed measurement of only 2 relevant enzymes.  The enzymes chosen were  cytoplasmic alpha-glycerophosphate dehydrogenase (a-GPDH)  and citrate  synthase.  Table Variate a-GPDH (unitsg  VII Pre-test Count 10 0.066±.024 5 0.058+.029  Post-test 0.085±.021 0.066±.011  P-value 0.58  protein)  Group Iron Placebo  Citrate synthase (unitsg protein)  Iron Placebo  10 5  0.048±.010 0.042±.012  0.76  1  1  0.047+.011 0.039±.017  Table VII: Descriptive statistics of enzyme activity, Iron group vs. placebo group.(1 unit = Ijimole of product produced under saturating conditions in 1 minute at 25° C)  There was not a significant difference between the two treatments with regards to either enzyme (table VII).  Probability values obtained from  ANOVAs were .58 for the change in a-GPDH and .76 for the change in citrate synthase.  34  Finally, it can be noted that three women who were on the placebo treatment became anemic (hemoglobin levels below 12.0 gdl" ). 1  The data  from these subjects was excluded from the MANOVA's but are presented (Appendix L) for descriptive purposes.  35  Discussion  Eight weeks of iron supplementation ((a daily dose of 100 mg elemental iron (320 mg FeSo4 taken as Slow Fe® twice a day, as tolerated)) to prelatent/latent  iron  deficient  physically  active  females  did not  significantly enhance work capacity nor the activity of 2 oxidative muscle enzymes (citrate synthase and a- GPDH).  Within the limitations of this  study the presence of a serum ferritin below 20 ng-ml" does not pose a 1  significant handicap to anaerobic or aerobic capacity.  Serum  Ferritin  From the 155 females involved in the initial screening, the mean serum ferritin was 29.9 ngml" for  Canadian  female distance  it was found that  which is similar to that reported  1  runners;  27.9 n g m l "  (Clement and  1  Asmundson, 1982) and U.S. cross country skiers; 32.8 ngml" al., 1986).  1  (Haymes et  A random sampling of 95 females (age 20 - 39) across Canada  found a mean serum ferritin of 23 n g m l " , but in 44 controls who were 1  selected on the basis of normal hemoglobin the mean ferritin value was 35 ngml"  1  present  Valberg et al., 1976). study  The distribution about the mean in the  is also similar to these  previous  studies in that the  majority of the females had serum ferritins below 30 ngml" (ie. 60% < 30 1  ngml" ). 1  Clement and Asmundson  skewedness to the distribution.  (1982) found  an even  greater  Using a criterion of 25 ngml" , 82% of the 1  36  females in their study were prelatent iron deficient. difference may be training volume.  The reason for this  The subjects in the present study were  mostly recreational runners while Clement and Asmundson studied elite level runners.  Magnusson et al. (1984) question the validity of low serum ferritin levels in distance runners.  They observed normal erythropoiesis and low serum  ferritin concentrations in a group of middle and long distance runners and it thus appeared that a lack of iron had not limited erythropoisis. Magnusson et al. concluded that a true iron deficiency did not exist. Instead, increased intravascular hemolysis, as evidenced by the formation of the  haptoglobin-hemoglobin  complex, causes a shift in red cell  catabolism from the reticuloendothelial system to the hepatocytes.  The  fact that 3 of the 18 placebo treated subjects in the present study became anemic is suggestive that at least in recreational female runners the presence of a low serum ferritin concentration does indicate a true iron deficiency.  Inadequate dietary iron intake appears to be a major contributing factor to the prevalency of iron deficiency.  Iron intakes of the prelatent/latent iron  deficient athletes who completed the 8 week study averaged 11.8 ± 3.7 mg/day during the pretest period and 11.0 ± 3.0 mg/day during the post test period (based on 3 day dietary records; Appendix G).  Approximately  80% of the females had intakes below the Canadian recommended intake of 14 mg/day (Health and Welfare Canada, 1983).  The Nutrition Canada  survey (1975) also showed inadequate iron intakes for the females in the  37  20 - 39 age category; ie. 11.1 mg/day (Health and Welfare Canada, 1975). British Columbians participating in this national survey were even lower in iron intakes with a mean of 10.0 mg/day.  These results reinforce the  common finding that it is difficult for the typical Western diet to meet the iron demands of the menstruating female (Clement and Sawchuck, 1984).  The only significant difference between the two treatment groups was the change in serum ferritin levels (Table III). serum ferritins of near 12.3 ngml" . 1  to 17.2 ngml"  1  Both groups started with  mean  The control group's mean level rose  while the iron group's rose to 37.7 n g m l " .  Although  1  statistically significant, this rise in serum ferritin is still modest when one considers the normal range extends to 160 ngml" .  Schoene et al.,  1  (1983), supplemented with a similar dosage (300 mg/day) but for only 2 weeks and found an increase in ferritin levels from 10.0 ± 2.0 to 22.1 ±7.8 ngml" . 1  In  the  present  study  it was  hoped  that  8 weeks  of  supplementation would be sufficient to bring the mean ferritin level to greater  than 60 n g m l " . 1  Ferritin levels below 64 n g m l "  indicate an iron deficient state (Heinrich et al., 1977).  1  may still  The inability of  this study to show statistically significant changes in work capacity was arguably  handicapped by the still sub optimal post treatment ferritin  levels.  Heinrich et al. (1977) correlated iron absorption with serum  ferritin concentration.  Diagnostic  5 9  Fe  + 2  absorption appeared to be a more  sensitive indicator of depleted iron stores. ferritin values up to 64 ngml" prelatent iron deficiency.  1  It was concluded that serum  could still be suspect of representing  Exhausted iron stores cannot be definitely  38  excluded as a possibility until serum ferritin concentration rises above this level.  Combining the data of Schoene et al. (1983) with the present  data it is apparent that replenishment follows an asymptotic curve.  A  linear extension of Schoene et al's data would result in a mean serum ferritin  level  of 60 ng-ml"  1  after  8 weeks.  Supplementation  of  prelatent/latent iron deficient females should ideally be continued for perhaps 16 weeks to ensure mean levels reach the 60 to 70 ng-ml" range. 1  The large standard deviation of post test serum ferritins for the iron supplemented group in both Schoene et al's (1983) study (22.1 ±7.8 ng-ml" ) 1  and the present research (37.7 ± 19.7 ng-ml" ) is noteworthy.  One  1  individual in this study increased their ferritin from 7 to 12 ng-ml"  1  while  another similarly iron treated subject responded with an increase from 18 to 83 ng-ml" . 1  There is thus a great deal of variability in how subjects  respond to iron therapy.  Differences in absorption are likely the largest  contributing factor to this variability. variability imposed by other factors.  Controls were in place to limit the These included pill counts at 4 and 8  weeks, checks for blood loss in urine and stool and standardized instructions for when and how the supplements should be taken.  The  absorptive mechanism of allowing the amount of iron crossing the small intestinal epithelial barrier to be related to internal iron status can be defective in many people (Ehn et al., 1980, Jacobs, 1985).  The problem is  probably genetic and/or due to interrelationships that iron has with other nutrients.  39  Considering this latter alternative, it is conceivable that multiple mineral deficiencies can occur. Attempts at repleting one mineral may not meet with success but instead could compound the imbalance of the internal system.  As a speculative example:  The phytates, oxalates and alkali that  inhibit iron absorption will also inhibit copper and zinc absorption (Kreg and Murray, 1986).  Copper, zinc and iron could be further synchronously  depleted due to their similar food sources (ie liver, meat).  A vegetarian  diet may not only contribute to iron deficiency but to copper and zinc deficiency.  Typically, only the iron deficiency is diagnosed and megadoses  of iron are prescribed.  Absorption of copper and zinc are both inhibited by  iron and these two minerals could be further depleted.  Copper is essential  for hemoglobin synthesis and iron mobilization, while zinc is a component of numerous enzymes.  A deficiency in either will likely affect iron  metabolism and absorption.  Support for this concept can be found in a  study on copper deficient swine (Lee et al., 1968).  Copper deficient pigs  developed a severe hypochromic, microcytic anemia and had low serum iron concentration.  In these pigs there was a defect in the transfer of iron  from the intestinal mucosa to the blood and from the reticuloendothelial cells to the plasma and also in the incorporation of iron into hemoglobin. The oxidation of F e constituent,  +2  by caeruloplasmin, of which copper is a prime  may explain these defects (Jacobs  and Worwood,  1974).  Although the above argument is speculative it is clear that a broad focus to the diagnosis of iron and other trace mineral deficiencies is necessary.  40  Hemoglobin Hemoglobin levels among the 155 females initially screened averaged 13.0 gdl  - 1  (Figure 2).  The normal range is defined as 12.0 to 16.0 g d l " . 1  Thirteen females had hemoglobin levels below this normal range and could be classified as anemic.  The observation that 12 of the thirteen had  ferritin levels below 30 ngml"  1  (13.6 ± 8.1 ng-ml" ) underlines the fact 1  that of the anemias iron deficiency anemia is the most prevalent in this group.  The change in hemoglobin values pre to post was negligible for both treatment groups (Tables I and III).  This is in agreement with Pate et al.  (1979) who found that oral iron supplements when administered to normal, non-anemic women athletes, have no statistically significant effects on hemoglobin levels. 14.38 to 14.96 gdl" elemental iron/day).  Pate et al. did note though a modest improvement from 1  over their treatment period (5 to 9 weeks with 50 mg There was thus some question prior to the present  study whether changing hemoglobin levels would influence our results.  If there was a difference between the treatment groups it would have confused the issue for a rise or fall in hemoglobin levels would affect oxygen carrying capacity and thereby affect work capacity (Haymes, 1973). Edgerton et al. (1972) suggests that the capacity for work is directly related to hemoglobin concentration.  To differentiate the effects of iron  depletion from hemoglobin levels it was essential that hemoglobin levels remained constant.  41  Total Iron, U.I.B.C., and % Transferrin Saturation These variables were added to the iron status measurements to help discriminate between prelatent and latent  iron deficiency.  They do not  reflect decreasing iron stores until the stores are exhausted.  A low serum  ferritin (< 20 ngml" ) coupled with an abnormally low total iron and % 1  transferrin saturation, high UIBC but normal hemoglobin levels would indicate the latent iron deficient stage.  It was hypothesized that a subtle  difference in work capacity may only become apparent when this subgroup is isolated.  Only 5 women of the iron treated group initially had latent iron deficiency. This was unfortunate from a scientific viewpoint in that the small 'n' makes a finding of statistical significance less likely. here.  Such was the case  From a single post hoc ANOVA on this subgroup vs. the placebo group  on the AST performance variable a significant difference could not be found.  Based on this datum latent iron deficiency does not affect work  capacity.  Pakarinen  (1980)  has criticized  transferrin saturation measurements. could be found in this study. variables (table VI).  the reliability  of total  iron and  Evidence supporting this criticism  Correlations were computed between all  Of interest would be whether serum ferritin levels  were correlated to the other parameters of iron status.  Only UIBC  exhibited strong correlations with serum ferritin levels on both the pre and post tests (r = -.33 and -.47 respectively, with associated p values of  42  p<.1 and p<.02).  The post test % transferrin saturation correlated well  with serum ferritin (r - .36; p<.1) but the pre test didn't, nor did the total iron values.  A low correlation  means that only a small portion of the  variance in one variable is predictable from the variance in the other. other words,  the  serum ferritin  prediction of the total iron.  concentration  does  In  not aid in the  These results are not totally unexpected.  In  healthy persons a weak inverse relationship was observed between plasma ferritin concentration and TIBC, but not the serum iron concentration or the transferrin saturation (Cook et al., 1974).  Jacobs et al. (1972) found  no correlation between serum ferritin concentration and % transferrin saturation in healthy males, although for females the correlation was significant (r= 0.66, p<.001).  This sex difference may be explained by the  larger number of women with low serum ferritins.  In both sexes in Jacob  et al's (1972) study, a serum ferritin concentration below 10 ngml" associated with a transferrin saturation of less than 16%. be said in the present study.  plasma  pool  1  It can be presumed that iron delivery to the  is almost certain to  between  The same can  also had a transferrin saturation  be impaired when  concentration drops below this 10 ngml" relationship  is  On the pretest measures six of the seven  subjects with ferritins below 10 ngml" below 16% (Appendix I).  1  serum  saturation is not well defined.  ferritin  1  level.  serum ferritin  Above this level the  concentration  and  transferrin  Also of interest would be whether the  change in serum ferritin pre to post was correlated to the change in the other iron status parameters. significant (r < 23; p>.2).  In this case none of the 3 correlations were  In many instances serum ferritin levels changed  independantly of changes in total iron, UIBC, and % transferrin saturation.  43  One, or a combination of 3 possible conclusions can be made; 1) Concurring with Pakarinin (1980) total iron and transferrin saturation are not good tests to show depleted iron stores.  Only serum ferritin is  reliable. 2) Serum ferritin measurements are unreliable.  Bannister and Hamilton  (1985) performed repeat tests on hemoglobin, total iron, TIBC and serum ferritin and found coefficients of variation which were 0.7%, 1.78%, 1.99%, and 10% respectively. 3) The pattern of depletion/repletion  has a high degree of individual  variability. All  three  possibilities  likely  contribute  in that  lability  of  blood  measurements is considerable and the two sets of data can behave independantly due to individual patterns of depletion/repletion.  Work Capacity None of the work capacity measurements improved significantly (Table IV).  The anaerobic speed test elicited the greatest difference between the  two  groups,  but statistically  conditions of this study  this  was not significant.  Under the  it can be concluded that the prelatent/latent  stage of iron deficiency does not pose a significant handicap to either aerobic or anaerobic performance.  Supporting this conclusion derived from  a MANOVA was the lack of a significant correlation between the change scores for serum ferritin and the work capacity variables (Table VI).  44  Unlike the iron status measurements, the work capacity tests showed good test-retest reliability as evidenced by the placebo groups fairly stable scores and  pre to post correlations (table VI).  The  least reliable  measurements were from the Wingate bicycle ergometer test (r = 0.39 for alactic power, r = 0.69 for lactacid power).  The other exercise tests had  test-retest correlations of over 0.78.  It is interesting to speculate on the fact that except for alactic power, the direction of change in the physical work capacity measurements was in favor of the iron supplemented group. limitations would suggest that further supplementation,  if  at  all  To view the results in light of the research is warranted.  advantageous,  would  most  Iron  likely  affect  oxidative energy production, needed in some degree in exercise tests of longer duration than that of alactic power.  The anaerobic speed test  showed  statistically  the greatest differentiation (albeit  between the two treatment groups (p=.21).  nonsignificant)  Although the contribution of  the aerobic pathways is small in the approximately 45 second effort of the AST, aerobic capacity is critical to the achieved performance.  If, as  hypothesized, iron supplementation enhances aerobic capacity, the build up of lactic acid is delayed and running time is extended.  Outside the focus of this study it was noted that % body fat correlated highly to AST performance (r=.67, p<.001).  Also noteworthy is the large  standard deviation (eg. 45.1 ± 16.9) associated with this test.  A more  homogeneous sample population or adjustment of treadmill speed to narrow the standard deviation may aid in it's discriminating ability.  45  A significant difference between the groups on the V0 max variable was 2  not evident (p=.31; table IV).  Work by Schoene et al. (1983), Finch et al.  (1976, 1979), Davies et al. (1982) and Vellar and Hermanson (1971) would not lead one to expect significant changes in this variable as long as hemoglobin levels stayed within the normal range.  In the study by Davies  et al. (1982), rats were raised on an iron deficient diet so that hemoglobin levels and muscle mitochondrial content were depressed. restored  in the  rat's  diet to  assess whether  Iron was then  V0 max 2  mitochondrial content better predicted exercise endurance. levels increased in parallel with V 0 m a x , 2  while  2  2  muscle  Hemoglobin  mitochondrial capacity  and running endurance improved in parallel but more slowly. suggest that V0 max is a function of 0  or  These results  transport whereas endurance is  more dependant on the ability of the mitochondria to consume oxygen.  This  can also be interpretted to mean that the mitochondrial capacity to consume 0  Although  is much greater than the ability of the circulation to supply 0 .  2  2  V 0 m a x was not expected to improve, peripheral changes (ie. 2  increased enzyme activity) had been hypothesized to improve endurance capacity.  The anaerobic threshold and maximal treadmill velocity during  the V 0 m a x test should reflect this improvement but once again the 2  change was  not statistically significant.  Endurance  significantly associated with serum ferritin levels.  capacity  is not  46  Enzyme  Activity  Measurement of enzyme activity changes represented the most objective evaluation as to the effects of prelatent/latent iron deficiency in this study.  No significant difference could be found in the two enzymes  measured (cytoplasmic alpha glycerophosphate dehydrogenase and citrate synthase). unitsg"  1  The mean activity of a-GPDH increased from .066 to .085 protein for the iron group and only .058 to .066 for the placebo  group but the associated p value of .58 does not tempt further speculation (see table VI).  Citrate synthase activity also showed a non significant  change over the treatment period for both groups.  It can be concluded that a more severe degree of iron deficiency is necessary to affect cytoplasmic a-GPDH and citrate synthase activity. The present study lends support to the theory that iron dependent enzyme activity drops in concert with a drop in hemoglobin and not before it. conclusion is in agreement with Celsing et al. (1986), who  This  studied  different enzymes but also concluded that it is possible to have intact endurance capacity and maximal enzyme activity despite diminished or depleted iron stores.  The NAD-linked form of a-GPDH was assayed instead of the mitochondrial form as assayed by Finch et al., (1976, 1979).  Their activities are  comparable as they both participate in the NADH-glycerol shuttle.  phosphate  The fact that work capacity was not significantly affected by the  prelatent/latent stage of iron deficiency makes it difficult to comment on  47  the debate over the relative importance of a-GPDH (Finch et al., 1976,1979 and McLane et al., 1981).  Conclusions Oral iron supplementation (320 mg ferrous sulfate = 100 mg elemental iron taken as Slow-Fe® twice a day for 8 weeks) was successful in raising serum ferritin (12.4 ± 4.5 to 37.7 ± 19.7 ngml" ) 1  in prelatent/latent iron  deficient females but was not associated with significant improvements in work capacity, nor the activity of 2 muscle enzymes, cytoplasmic a-GPDH and citrate synthase.  48  Recommendations for future study 1.  Treatment should induce higher serum ferritin response.  Consider  longer treatments and/or higher dosages. 2.  Improve the discriminating ability of work capacity measurements.  For  example, consider familiarization tests to determine the ideal treadmill speed for each subject during the AST (ie. to bring exhaustion between 35 - 55 seconds). 3.  Examine  factors  to  iron  5. Select a larger group of latent (as opposed to prelatent/latent)  iron  supplementation. 4.  leading  to  the variable  response  For example multiple mineral deficiency.  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Torun, "Anemia and physical work capacity", Clinical Haematology. 3: 609-626, 1974. Volpicelli, N.A., "Effect of sports on the gastrointestinal tract and liver", Sports Medicine: Fitness. Training. Injuries. (O. Appenzeller and A T . Kinson, eds.), 2 ed., Urband and Schwarzenberg, Baltimore, 1983, pp. 91-98. n d  Weib, M., T. Krauter, K.H. Graff, W. Abmayr and W. Weicker, "Iron pathway and iron loss in different forms of training and the origin of sports anemia", International Journal of Sports Medicine. 4: 64, 1983. Williamson, M.R., "Anemia in runners and other athletes", Physician and Sports Medicine. 9(6): 73-79, 1981. Winick, M., "Beating iron deficiency anemia: A family physicians guide", Modern Medicine of Canada. 36(11): 1571-1581, 1981. Wirth, J.C., T.G. Lohman, J.P. Avallone Jr., T. Shire and R.A. Boileau, "The effect of physical training on the serum iron levels of college-age women", Medicine and Science in Sports. 10(3): 223-226, 1978. Wishnitzer, R., E. Vorst and A. Berrebi, "Bone marrow iron depression in competitive distance runners", International Journal of Sports Medicine. 4: 27-30, 1983. Wood, M. and P. Elwood, "Symptoms of iron deficiency anemia. A community survey", British Journal of preventive and social medicine . 20: 117,1966. Yoshimura, H., "Anemia during physical training (sports anemia)", Nutrition Reviews. 28 (10): 251-253, 1970.  65 Appendix A  Review of Literature  OVERVIEW OF IRON METABOLISM  Iron is a trace element found in all cells of the body and is essential for life.  In fact, virtually all living matter must procure  a certain amount of iron for normal development (Finch, 1980). Many plants and animals have developed mechanisms for acquiring this crucial element. oxygen transport  The requirement of a hemoglobin/myoglobin  system expands  Man's requirements  compared to animals without this transport system.  tenfold  This fact and  the progressive change in diet away from meats that began with the cultivation of grains about 10,000 years ago has made the maintenance of proper iron balance difficult.  The 3-5 g of total body iron in the adult allows many key biochemical reactions to take place.  It is responsible for electron  transport  of oxygen  (cytochromes),  oxygenases)  activation  (oxidases and  and for the transport of oxygen and carbon dioxide  (hemoglobin and myoglobin).  Hemoglobin accounts for about 2.5 gs  (2500 mg) of the 4 g total; myoglobin values are approximately 150 mg and the metalloenzymes account for 6-8 mg.  There is  66 about 3 mg of iron in the plasma and this is bound to a carrier protein, transferrin.  Tissues and organs such as the liver, spleen,  and bone marrow store iron as ferritin, or the more insoluble hemosiderin, in varying amounts (Haymes, 1973, Valberg, 1980).  Measurement of the degree of saturation of transferrin (and red cell  protoporphyrin)  can detect  iron  deficient  erythropoiesis,  while quantitation of iron in plasma ferritin and in bone marrow reveals the status of the body's stores (Sonnenwirth and Jarett, 1980).  Stores in adult men may be 500 to 1000 mg while the iron  stores of women seldom exceed 500 mg (Bothwell et al., 1979).  Internal exchange of iron is accomplished by the plasma, B-glycoprotein, transferrin (Finch, 1980).  Normally transferrin is  about 35% saturated with iron and the normal plasma iron level is about 130 ug-dl" (23 umolT ) in men and 110 ugdl" 1  in women.  1  1  (19 umoll" ) 1  There is a continuous reutilization of iron catabolized  in the body.  Most of this is in red blood cells which circulate for  approximately  120 days  before  reticuloendothelial system, which marrow, and spleen.  being  catabolized  by  is comprised of liver,  the bone  Iron absorbed from the small intestine or  from hemoglobin in the reticuloendothelial system is taken up by transferrin and is transported to the bone marrow for formation of red blood cells.  The recycling of iron in the red cell mass helps  maintain iron balance. of iron metabolism.  The following diagram shows the pathways  67  Dietary Iron (14±4 mg/day: about 6 mg/IOOO kcal)  I Intestinal Mucosa  (Absorption about 1 mg/day)  I  Plasma Iron '(pool about 3 mg: turnover about 10X/day)^  Erythroid Marrow (uptake about 25 mg/day) Circulating Erythrocytes (pool about 210Omg: daily turnover 18 mg)  Interstitial Fluid  I  Parenchymal ExchangeEspecially Liver (about 6 mg/day)  I  Ferritin Stores Reticuloendothelium (25 mg/day from erythron)  Figure #1. Pathways of iron metabolism in man (excretion omitted). (From Finch, 1980).  68  The most remarkable feature of iron metabolism in humans is the degree to which the body store is conserved. For normal men only 1 mg/day is lost. Two thirds of this iron is excreted from the gastrointestinal tract as extravasated red blood cells, iron in bile, and iron in exfoliated mucosal cells. The other third is accounted for by small amounts of iron in sweat, desquamated skin, and in the urine (Bothwell et al., 1979).  Iron losses in the male vary less than in the female. About 0.5 mg may be lost in the iron deficient male and as much as 1.5 to 2 mg/day can be lost when excessive iron is consumed (Bothwell et al., 1979).  Additional menstruation.  losses  of  iron  occur  in  the  female  due  to  Menstrual losses average 0.5 mg/day although 10%  of the normal menstruating females lose over 2 mg/day.  Estrogen  containing oral contraceptives reduce menstrual loss by about one-half,  while  intra-uterine  devices  increase  losses  (Finch,1980).  Athletes  may  increased losses.  have  increased requirements for  iron  and  Endurance athletes, especially females in their  reproductive years, are at a high risk of becoming iron deficient. In other words, the average amount of iron derived from their diet may be less than the losses from their body.  69 A negative iron balance would cause a depletion of iron stores, increased  absorption  concentration, microcytic  from  the  impairment  of  hypochromic  gut,  a  fall  in  erythropoieses,  anemia (Winick,  1981).  however come into balance at any of these stages.  plasma  iron  and  finally  One  could,  The stage at  which iron deficiency affects work capacity has not been fully elucidated nor has the biochemical locus of this detriment been determined (Strauss, 1983).  This review areas: 2)  1)  of iron metabolism will address two  pertinent  the causes for a negative iron balance in athletes; and,  the effects this may have on work capacity, especially in the  prelatent/latent iron deficiency stages.  NEGATIVE IRON BALANCE IN ATHLETES  In endurance athletes, the sites of jeopardized iron balance may be inadequate dietary intake, malabsorption from the gut, or increased iron losses in the sweat, urine or feces.  70 Inadequate Dietary Intake  It is difficult for the typical western diet to meet the iron demands of the menstruating female.  The average western diet  supplies only 5-7 mg of the iron per 1000 Kcal.  Since the  recommended dietary intake in Canada for females is 14 mg based on an average 10% absorption (Health & Welfare Canada, 1983; ) or even  18 mg as  is the  U.S.  Recommended Dietary  Intake  (Committee on Dietary Allowances, 1980), intakes below 2000 Kcal  are  often  associated with  Sawchuk, 1984).  iron  deficiency  (Clement  &  In fact, nutritional analysis of female distance  runners revealed an average intake of 12.5 mg (Clement and Asmundson, 1982).  Female athletes in sports such as gymnastics,  ballet, and distance running, where low body fat is an asset, run a high risk of developing iron deficiency due to a low caloric intake.  Much of the iron in food is absorbed  inefficiently.  The  absorption of iron depends on both the amount and bioavailability of dietary iron.  The bioavailability of food iron is quite varied.  Heme iron, which is in the reduced, ferrous (Fe ) form and is +2  found in meats, is easily absorbed; (i.e. 10-37% of intake is absorbed)  (Bothwell et al., 1979; Finch, 1980; Hallberg, 1984).  Non-heme iron, which represents by far the largest amount of dietary  iron (i.e. vegetable foods),  has poor bioavailability as  usually less than 10% of intake is absorbed (Bothwell et al., 1979;  71 Finch, 1980).  In vegetable foods, ferric iron (Fe ) is bound in +3  insoluble complexes and its' absorption is altered to a great extent by the other ingredients of the meal (Passmore and Eastwood, 1986).  Meat, apart from supplying heme iron, appears to promote  the absorption of non-heme iron, probably production of gastic acid (Finch, 1980).  by meat stimulating the  Most of the non-heme iron  ingested is in the ferric form and must be reduced to the ferrous state to be easily absorbed.  The acid secretions of the stomach  aid in this conversion, hence absorption is mainly from the upper portion of the small intestine. reduces  Fe  + 3  to F e . + 2  Ascorbic acid (Vitamin C) also  Antacids negate this effect.  Interfering  with the absorption of non-heme iron is phytic acid found in cereals.  Phytates, carbonates, phosphates, tannins and oxalates  react with iron to form insoluble compounds in the intestine (Ganong, 1981; Passmore and Eastwood, 1986). counteract  the effect of excess  phosphorus  Calcium may  for  in sufficient  amounts calcium will combine with phosphates and free iron for use (Kirschmann, 1984)  ^  The high prevalence of iron deficiency is largely a function of the limited availability of iron in the contemporary diet (Bothwell et al., 1979).  Paleolithic man had a diet with meat comprising up  to 8 0 % of the total diet (Aronson, 1985). protein  were  ample.  The  agricultural  reduced animal protein consumption.  Iron, folic acid, and revolution  drastically  72 Malabsorption  The limited physiological losses of iron point to the primary importance of absorption as the determinant of the body's content of iron.  The major regulator of iron repletion is that of keeping the absorptive 1973).  processes attuned to body  requirements  (Haymes,  Thus the amount of iron crossing the small intestinal  epithelial barrier is related to the internal iron status.  The extent  to which this mechanism operates in a normal population is quite variable since there is a wide range in the amount of storage iron found in individual subjects (Jacobs, 1985).  Absorption  may  be through  passive  diffusion  and  active  transport, although most iron is passed through the mucosal cells by binding to the protein apoferritin, forming ferritin molecules. If there is a depletion of the body's  iron stores there is a  compensatory increase in the amount of iron absorbed into the blood.  The rate at which iron enters the blood is dependent upon  the quantity of ferritin present in the intestinal mucosal cells, which in turn is dependent on the amount and saturation of transferrin.  The ferritin level in the mucosal cells will decrease  in the event of an iron deficiency which will consequently allow more iron to enter the plasma (Klausner et al.,1985).  73  Mucosal Phase  Luminal Phase  Blood  Food  ^/\\  Digestive/ J . / u  i  i c e s  J  Non-heme Compounds  \ \  i •. %  \ Heme  v ligands  ^^Available ^^•Complex  1  Unavailable Complex  n r m r  ^  ^* i Transport ! Protein s  l  %;  —•Transferrin  Noil '|1 Ferritin!\  Figure #2. The effects of various intraluminal factors on the absorption of heme and non-heme iron. (From Bothwell, 1979).  74  In spite of this mechanism for  maintaining iron balance,  absorption disturbances appear to contribute to a negative balance in a number of athletes.  Ehn et al., (1980), using radioactive iron,  found that absorption in iron deficient runners was 16.4%, while the iron deficient controls absorbed 3 0 % of the labelled iron. Similar discrepancies between the absorption values of athletes versus  non-exercisers  have been reported by Clement et al.,  (1983), Heinrich, (1970), Heinrich et al., (1977), and Bannister and Hamilton,  (1985).  The  mechanism  disturbances has not been determined. of  intestinal  hurry  can  be  for  these  absorption  It is known that any cause  associated with  iron  deficiency  (Passmore and Eastwood, 1986), but whether this is a factor in athletes is not known.  Bannister and Hamilton, (1985), proposed  that heavy training causes the plasma transferrin saturation to be elevated.  This would retard the release of iron from the mucosal  cells of the intestine.  Not until periods of relative rest, such as  peaking, would the % saturation of transferrin decline, allowing iron to be absorbed, and the RBC count and Hb concentration to increase.  75  Gastrointestinal Blood Loss  Long distance running may cause gastrointestinal bleeding that contributes  to iron deficiency  in some runners  Potera, 1984; Stewart et al., 1984).  (Porter, 1983;  Four recent studies have  shown blood loss through the stool after long distance races (10 km to marathon) (McMahon et al. 1984; Porter, 1983; Stewart et al., 1984; Volpicelli, 1983).  Using the Hemoccult test to detect gastrointestinal bleeding after racing, Porter (1983) found an incidence of 8%, Volpicelli (1983) 2 0 % and McMahon et al. (1984) 22%.  Stewart  and  colleagues used the Hemoquant assay (a more sensitive assay, yet not at present commercially available) and found nearly 30% of stool specimens collected after a race contained more than 3 mg of hemoglobin per gram of stool.  In Stewart et al.'s (1984) study,  the mean fecal hemoglobin rose from 0.99 mgg" to 3.96 mgg"  1  1  before the race  after racing, with 20 of the 24 runners showing  increased levels (p < 0.01).  Seven of these runners had equivalent  iron losses of more than 2 mg.  The significance of gastrointestinal bleeding depends on the quantity of blood loss and the frequency of occurrence (Buckman, 1984).  It appears that bleeding is associated with the degree of  76 running  stress.  relationship  McMahon  between  et  bleeding  al. (1984) and  noted  effort.  a  possible  Gastrointestinal  bleeding may thus be a phenomenon of distance racing or very strenuous training and not distance training per se (Eicher, 1985). In most runners, the bleeding resolves within 72 hours (McMahon, 1984).  Exercise associated gastrointestinal bleeding remains to  be, though, a potential contributor to iron deficiency (Buckman, 1984).  The cause of running induced gastrointestinal blood loss is  obscure but is speculated as being due to transient gut ischemia (Porter, jarring  1983; Potera, 1984).  and/or  a repetitive  effect on the intra-abdominal organs  Porter, 1983).  traumatic  (Buckman, 1984;  Causes of gastrointestinal bleeding not associated  with exercise and their relative frequency can be obtained from a retrospective study on 371 patients with iron deficiency anemia (Bannerman  et al., 1964):  gastrointestinal hemorrhage 60%,  hemorrhoids 10%, salicylate ingestion 8%, hiatus hernia 7%, peptic ulceration 7%, diverticulosis 4%, neoplasm 2%, ulcerative colitis 2%, and miscellaneous causes in 4%.  No explanation for the occult  gastrointestinal blood loss was found in 16%.  In adult males and post-menopausal females, iron deficiency is nearly always due to gastrointestinal bleeding (Bothwell et al., 1979). negative  A daily blood loss of 7-10 ml is sufficient in causing a iron  balance.  Unless  the  bleeding  is caused  by  hemorrhoids, such bleeding will usually escape notice by the patient.  77  Acetylsalicylic acid is a common drug known to cause both acute and chronic Corticosteroids, non-steroidal  blood loss from the gastrointestinal  phenylbutazone,  indomethacin  anti-inflammatory agents  also cause gastrointestinal bleeding. usually  parallels dosage.  tract.  and  other  and anticoagulants  can  Blood loss due to aspirin  Typical losses may be 0.5 ml daily,  while patients who take acetylsalicylic acid regularly may lose 2 ml/day and even larger doses (2-6 tablets daily) could result in daily blood losses of more than 5 ml.  Salicylates do their damage  through increased exfoliation of the gastric mucosal cells, erosive or hemorrhagic gastritis, and inhibition of platelet which may prolong bleeding.  aggregation  The variety of compound preparations  containing acetylsalicylic acid and the frequency in which athletes ingest these and other anti-inflammatory medications makes it apparent that drug  use is an important consideration  in the  diagnosis of iron deficiency in athletes.  Increased fecal iron loss could also be due to an elevated secretion of endogenous iron in the bile.  This explanation was  offered by Ruckman and Sherman (1981) in noting increased fecal iron losses in exercising rats.  78  Iron Loss in Urine  The observation of increased hemolysis with exercise has been made by a number of researchers (Davidson, 1964; Ekblom et al., 1977; Lindemann et al., 1978; Puhl and Runyan, 1980; Shiraki et aL, 1974; Yoshimura, 1970; Dufaux et al., 1981; Ehn et al., 1980; Eichner, 1985; Hunding et al., 1981; Magnusson, 1984; and Porter, 1981).  A low serum haptoglobin indicates increased intravascular  hemolysis.  It  is debatable whether  increased iron loss.  this contributes  to  an  Hallburg and Magnusson (1984) did not see an  increased iron loss in urine suggesting that hemolysis was slight and that the excretion  of heme iron was  prevented  formation of the haptoglobin-hemoglobin complex. prevents excretion of free hemoglobin in the urine.  by  the  This complex The complex is  then taken up by hepatocytes and the iron is recycled.  Others (Clement and Sawchuk, 1984; Davidson, 1964, 1969; Siegal et al. 1979; Dufaux, 1981; Eichner, 1985; Hunding et al., 1981; and Porter, 1981) would argue that hemolysis (possibly due to the traumas of increased circulatory  rate, increased body  temperature, compression of red blood cells by muscular activity or with foot strike in activities such as running, or acute exercise acidosis) causes excessive amounts of free circulating hemoglobin which can be excreted  in the urine.  This trauma could be  79 compounded in exercise as elevated levels of catecholamines increase both the osmotic and mechanical fragility of erythrocytes (Puhl and Runyan, 1980; Yoshimura, 1970).  Hunding et al. (1981)  calculated that the amount of free hemoglobin found in the plasma of some of their studies' competition runners was large enough (if not balanced by diet) to effect an iron loss that would eventually (over months) lead to iron deficiency and anemia.  Eichner (1985) did follow the hematological adaptations over eighteen months of a healthy long distance runner.  He concluded  that hemolysis, probably involving small numbers of the oldest red cells, does occur, and even this low-grade  runner's  could create and sustain a negative iron balance. with the above  theories,  Eichner  found the  hemolysis  In accordance  hemoglobin  and  haptoglobin levels would increase when mileage was decreased.  There is also a possibility that red blood cells could be released in the urine following physical exertion as a result of bladder trauma or bleeding from the kidney pelvis. present  as  grossly  red  urine  or  occur  This may be  microscopically  and  undetected (Blacklock, 1977; Boileau et al., 1980; Hayashi et al., 1980).  80  Iron Loss in Sweat  There is a relatively minute amount of iron in sweat, but with profuse sweating some researcers believe this is a plausible route for excessive iron losses in endurance athletes.  Vellar, (1968),  reported an iron content in sweat of up to 40 ug-100 ml" . As the 1  sweat production during one day of endurance running may well amount to 1-3 litres, an additional iron loss of 0.4-1.0 mg/day is possible, thereby potentially doubling one's iron loss (Clement and Asmundson, 1982).  More recent work by Brune et al. (1986) using a  very careful cleaning procedure and repeated consecutive sampling periods of sweat in the sauna found that iron losses in sweat are much lower.  Steady state sweat content was only 22.5 u,g iron T  1  sweat and this should have marginal effects on total body iron losses.  81  IRON DEFICIENCY WITH ANEMIA  The  most  apparent  physiological  consequences  deficiency are those that can be attributed to anemia.  of  iron  The criteria  for anemia in females has been set at hemoglobin (Hb) levels below 12 gdl" (Anderson and Barkve, 1970). 1  In males, the cut-off  value for anemia is 13.0 or 14.0 g d l " (Bothwell et al., 1979). 1  Hemoglobin, carried in the red blood cells, transfers oxygen from the lungs to the tissues so that oxidative reactions can take place in the cells.  From the cells, hemoglobin carries C 0 to the lungs to 2  be exhaled.  Both delivery of 0  reduced in an anemic individual.  2  and clearance of C 0 can be 2  Ohira et al. (1983) noted that  although C 0 is twenty times more soluble than 0 , the red blood 2  cell  2  is essential for carrying  approximately 90% of the C 0  2  or converting  to bicarbonate,  An inability to adequately clear C 0  2  from the working muscle because of a reduced Hb level or red blood cell count could cause a reduced muscle pH, which would limit the metabolic processes of skeletal muscle.  One of the major effects of anemia is on the circulatory system where  a greatly  (Anderson  increased workload is placed on the heart  and Barkve, 1970).  The viscosity of the blood is  dependent upon the concentration of red blood cells. viscosity is about three times that of water.  Normal blood  With severe anemia  82 the blood viscosity may drop to as low as one and one-half times that of water.  This causes a decreased peripheral resistance so  that far greater than normal quantities of blood are returned to the heart.  Cardiac output may increase two or more times than  normal (Guyton, 1977).  The increased cardiac output allows many  of the symptoms of anemia to go unnoticed at rest (Hallberg, 1978).  Most individuals with mild iron deficiency anemia have no  complaints that are dramatic enough to make them seek medical attention (Dallman, 1982).  With exercise, though, the heart is not  capable of pumping much greater quantities of blood than it is already pumping.  Tissue hypoxia thus develops as circulation  cannot meet oxygen demands. (Guyton,  1977),  impairment an  although  less  in work capacity.  elevated  heart  rate  Acute cardiac failure may ensue dramatic  outcomes  are  an  Work capacity is also reflected in  response  during  submaximal  work,  increased post exercise lactate levels, and a prolonged length of recovery (Anderson and Barkve, 1970).  There  is  a  significant  correlation  between  hemoglobin  concentration and work capacity (Anderson and Barkve, 1970; Dallman, 1982; Edgerton et al., 1972; Gardner et al., 1977; Ohira et al. 1983; Viteri and Torun, 1974).  The greater the severity of  anemia in humans and rats, the greater the decrement in work capacity (Ohira et al., 1983).  The hemoglobin levels need not  change markedly before there is a significant change in physical work tolerance (Gardner et al., 1977).  83 In studying female Sri Lankan tea estate labourers with varying hemoglobin levels, Gardner (1977) found that subjects with Hb concentrations between 11.0 and 11.9 gdl" a 2 0 % decrease  in their work  1  tolerance  showed approximately on an  incremental  treadmill test compared to those subjects with Hb concentrations above 13 g d l " . 1  The continuing parallel between Hb levels and  work capacity can be seen with higher than normal Hb levels. Ekblom et al. (1972) and Edgerton et al. (1972) have shown that work performance can be immediately improved when the oxygen carrying capacity is simply increased through blood transfusion. Ekblom's data indicated that work performance as measured by V 0  2  max could be improved if the Hb levels were increased above the 13.0 g d l  -1  level through reinfusion.  level below 14 gdl" suboptimal.  1  Parr (1984) considers any Hb  in females and under 16 gdl"  1  in males to be  The hematocrit for trained athletes may not be truly  ideal for maximum oxygen transport.  Heart rate response during moderate work was examined during the incremental treadmill test in Gardner's (1977) study.  There  was an approximate 4.7% elevation in heart rate with each 1 gdl" decrease in Hb.  1  Charlton et al. (1977) likewise showed the same  relationship between Hb and heart rate during exercise.  Post exercise lactate levels are also directly related to the degree of anemia, which indicates the degree of reliance on the anaerobic metabolic pathways.  Compensating for this is increased  84  levels of 2-3 diphosphoglycerate in the red blood cells which enhances oxygen release to the tissues (i.e. the Hb-02 dissociation curve shifts to the right) (Gardner et al., 1977; Charlton et al., 1977).  The length of recovery is prolonged with anemia.  Anderson and  Balkve (1970) reported that with manipulated Hb through repeated phlebotomy, a drop of 1.5 g d l "  1  extended the time to restore  resting cardiorespiratory functions by 10%.  The data on work capacity and iron deficiency anemia can be summarized with the statement than any decrement in function will be proportional to the degree of anemia.  85  IRON DEFICIENCY WITHOUT ANEMIA  In the past there has been a pre-occupation with anemia in the study  of iron deficiency.  deficiency picture.  Anemia is just part  of the iron  This portion of the review will first establish  the non-hematological effects of iron deficiency and then address the central question as to where in the stages of iron deficiency these tissue abnormalities occur.  The negative health effects of iron deficiency anemia can be partly ascribed to anemia and the impaired delivery of oxygen to the working muscles and partly to a deficiency of iron containing compounds, especially enzymes, in various sites (Dallman et al., 1978).  A wide range of tissue abnormalities have been described  in both iron deficient patients and experimental animals.  The  general assumption is that these occur as a result of defective synthesis of iron enzymes.  The total iron content of tissue  compounds is small but their distribution and specific metabolic functions are large.  Heme iron compounds in muscles include  myoglobin, the cytochromes, catalase and peroxidase.  Cytochrome  a, b, and c are located within mitochondria and are responsible for the oxidative production of energy (ATP).  Other cytochromes are  present in the membranous structure of the endoplasmic reticulum  86 and these assist functions such as oxidative degradation of drugs and endogenous substances (cytochrome protein synthesis (cytochrome b5).  P450 in the liver) or  Non heme iron compounds  include iron-sulphur proteins of the electron transport system in the mitochondria.  Examples of these are NADH dehydrogenase,  succinic dehydrogenase  and xanthine oxidase.  There are also  enzymes such as aconitase of the tricarboxylic acid cycle and tryptophan pyrrolase which do not contain iron but require the metal as a cofactor.  With iron therapy to iron deficient, anemic humans and rats, Ohira (1978,79) noted an improvement in physical work capacity and a reduction  in heart rate at a given workload before a  significant rise in hemoglobin, suggesting an effect unrelated to the elevation in hemoglobin that normally occurs.  Finch et al., (1976, 1979), Davies et al., (1982), Mackler, (1984) and McLane et al., ((1981) have proven that in rats iron deficiency results in defective oxidative energy (1976)  were  able  to  dissociate  metabolism. Finch et al.  the  effects  of  tissue  deficiency on work performance from those of anemia. deficient  rats  had  their  transfusion of erythrocytes.  anemia  corrected  with  iron Iron  exchange  Even with their anemia corrected  these animals had an impaired work performance.  Davies et al.  (1982), using a similar protocol, found that the recovery of the diminished V 0  2  max paralleled the rate of reversal of the anemia  87 but the recovery of endurance lagged behind, supposedly following the repair of muscle oxidative capacity.  Davies et al. (1982) and  Dallman (1982) thus hypothesized that the capacity to deliver 0  2  is closely related to the peak workload that can be attained in a brief, intense exercise ( V 0 max). 2  On the other hand, the capacity  to utilize 0 for oxidative phosphorylation is critical to the ability 2  to perform submaximal exercise for prolonged periods (endurance).  In an attempt to pinpoint the biochemical locus for this impairment to endurance, Finch et al. (1976) monitored both running ability and various enzyme activities as iron deficient rats underwent iron therapy. as  though  (a-GPDH)  In the rats Finch studied, it appeared  mitochondrial a-glycerophosphate  dehydrogenase  may be the limiting factor to performance as iron  therapy brought parallel increases in the rate of phosphorylation with a-glycerophosphate as performance.  substrate and the recovery in work  The effect of lowered  a-GPDH is two-fold:  one,  involving oxidative phsophorylation within the mitochondria and the  other a coupling with cytoplasmic NADH to  cytoplasmic NAD.  regenerate  Excess lactate could result from conversion of  pyruvate to lactate as a means of regenerating NAD and could also come  from  impaired  accumulation of pyruvate.  mitochondrial  phosphorylation  with  Lactic acidosis causes impairment of  muscle work (Finch et al., 1979).  88 Myoglobin and the cytochromes were excluded as a cause of muscle  dysfunction  by  Finch  et  al.  (1976)  because  their  concentrations, although decreased, did not improve with iron therapy during the time required to normalize running. investigators do not agree with this interpretation.  Other  McLane et al.  (1981) re-evaluated Finch's studies, questioning in particular the role of  a-GPDH  and the apparently  negligible effects that  depressed respiratory compounds myoglobin and the cytochromes had on fatigue resistance. According to Mclane et al., a-GPDH activity  is  secondary  to  the  malate-aspartate  shuttle  transporting reducing equivalents into the mitochondria. did  not  support  dehydrogenase  the  is  mammalian muscle.  a  hypothesis key  enzyme  that of  in  Results  a-glycerophosphate energy  metabolism in  Instead Mclane et al. (1981) concluded that,  "iron deficiency results in a decrease in mitochondrial enzymes in skeletal muscle with a decline in the maximum ability to utilize oxygen.  This reduction in the capacity for aerobic metabolism is  associated with an increased susceptibility to fatigue as a more rapid decline in contractile force and a greater increase in lactate concentration during contractile activities."  The search for a biochemical locus of a rate limiting step to work capacity in iron deficiency flounders due to the complexity of iron's effects.  Isolated iron enzyme defects can affect various  tissues and there is no consistent pattern thoughout the body (Jacobs, 1977).  Blayney et al. (1976) showed that in simultaneous  89 studies of myocardium and liver in iron deficient rats there was a depletion of myocardial cytochromes (and thus impaired activity thoughout the electron transport pathway), but in the liver only succinate-cytochrome  reductase  activity  skeletal muscle, myoglobin concentration one-half  though  the  proportional  was  affected.  may be reduced  reduction  of  In by  myocardial  myoglobin is much less (Dallman and Schwartz, 1965; Hagler et al., 1981).  Similarly, in skeletal muscle the cytochromes  become  more depleted than in cardiac muscle which has an increased obligatory workload with anemia (Dallman, 1978).  Within individual tissue and subcellular compartments, some compounds are affected far more than others.  In the  parenchymal cell, the mitochondrial cytochromes  are  more  than  the  cytochromes  in  the  endoplasmic  liver  affected reticulum,  cytochrome P450 is more readily depleted than cytochrome b5 (Dallman, 1978). only  between  There is a preferential utilization of iron, not  tissues  but within  tissues  and tissue  specific  adaptive responses to iron deficiency (Hagler et al., 1981).  The  identification of a rate limiting step in the skeletal muscle may be dependent upon the intensity required of the muscle, making the locus fiber type specific (Mackler et al., 1984; Mclane et al. 1981). Mackler et al. (1984) found the oxidative energy production by mitochondria  from  red  and  intermediate  muscle  from  iron  deficient rats to be markedly reduced, with production of ATP greatly  decreased.  According to McLane et al. (1981), it is the  90 intermediate (red, fast-twitch) affected.  The  muscle  a-glycerophosphate  fibers which are most shuttle is not affected in  white muscle although ATP formation by NADH and succinate is markedly  reduced  (Mackler et al., 1984).  The  workload  of  individual tissues is an important factor in determining where the effect of iron deficiency is greatest.  Also important is the rate of  tissue growth, turnover of specific iron compounds, time of onset during development and the degree of anemia (Dallman, 1978). These factors may explain why some tissues and iron compounds are vulnerable resistant.  to  iron deficiency,  whereas  others  are  quite  It also explains why a range of exercise tests is  warranted in studying the effects of tissue level iron deficiency.  Apart from a drop in work capacity through a depletion of certain enzymes, iron deficiency will affect cell morphology and growth which addition,  may also compromise exercise  iron deficiency  can affect behavior,  tolerance.  In  catecholamines,  thermogenesis (Dallman, 1982), cellular immunity and intestinal function (Siimes et al., 1980).  It  is  important  to  determine  the  significance  of  prelatent/latent iron deficiency in order to evaluate the extent to which priority.  its detection, treatment and prevention  deserve  a high  The significance of low iron stores in the non anemic  individual was previously believed to be solely a predisposition of the individual to a more severe anemia.  This alone has relevance  91 to athletes who may have an increased iron requirement.  Without  stores, a sudden loss of blood cannot be replaced at a rate greater than 3 mg iron per day, which can be obtained from the diet.  With  adequate stores, though, the replacement rate may be 25-50 mg iron per day (Bothwell et al., 1979).  The vulnerability of the  situation is illustrated by the fact that women who have no iron stores cannot go through pregnancy without becoming anemic (Bothwell et al., 1979).  The  hypothesis that prelatent/latent  iron deficiency affects  performance is dependent on establishing the sequential changes in the development of iron deficiency.  Stage  Serum T o t a l f e r r i t i n Iron  UIBC  T r a n s f e r r i r HemoSaturation qlobin  Normal  (np/ml) (ug/dl) (ug/dl) <*) 20-55 20-160 40-130 105282  Stage 1:  1-20  40-130  1-20  <60  1-20  <40  Iron depletion from stores Stage 2:  Iron-deficient erythropoisis Stage 3:  Iron-deficiency anemia  (q/dl) 12-16  20-55  12-16  >280  <20  12-16  >280  <20  <12  105282  Figure #3 Sequential changes (top to bottom) in the development of iron deficiency. (Values for females.)  92  It can be seen that iron restriction first affects iron stores, then transferrin saturation and lastly hemoglobin production.  Not  shown in the preceding diagram is the stage at which iron loss affects tissue enzymes or myoglobin.  Clouding the issue of iron status evaluation are the limitations of the commonly used indices.  The diagnosis of iron deficiency is  based on indirect methods of measurement. the individual variables  (eg.  The wide variation in  ferritin, serum iron, transferrin  saturation, bone marrow iron) even in apparently healthy persons, combined with the fact that they form part of kinetic systems which are influenced by many factors other than the availability of iron, means that definitions of the term iron deficiency based on these variables must be taken with considerable caution (Ericsson, 1970).  For example, physiological variation in the concentration  of hemoglobin is so great that only about half of the individuals with iron deficient erythropoiesis are identified by recognizable anemia (Cook et al., 1976). iron  deficient  rats,  the  As well, Finch (1979) notes that with dissociation  between  pre-exchange  hemoglobin concentration and work performance indicates that hemoglobin concentration per se is an unreliable indicator of the severity of tissue iron deficiency.  Serum ferritin measurements have also been put in question. At the high end of the scale, serum ferritin levels can be elevated  93 in clinical situations not related to iron stores. include  inflamation, chronic  liver  disease,  These situations and  malignancy.  Magnusson et al. (1984) re-evaluated the interpretation of reduced serum ferritin values in runners.  Their theory was that athletes  are not truly iron deficient, but that there is a shift in red cell catabolism hepatocytes.  from  the  reticuloendothelial  system  to  the  The hemoglobin-haptoglobin complex formed due to  intravascular hemolysis is taken up by hepatocytes. their argument was the finding  in their study  The basis of that  although  hematocrit, serum iron, transferrin saturation and serum ferritin values were low in athletes compared to controls;  sideroblast  counts, red cell indices and red cell protoporphyrin were normal, indicating that lack of iron had not limited erythropoiesis. confirm this theory a measurement of hepatocyte  iron  To (liver  biopsy) would be necessary.  The present investigation has a direct bearing on Magnusson et al's theory.  If iron supplementation had improved performance of  prelatent/latent iron deficient athletes, it would indicate a true iron deficiency existed.  A significant difference in performance  could not be found and by this measure Magnusson et al's theory could not be disproven.  On the other hand, the fact that 3 of the 21  placebo treated subjects dropped into anemia is indicative of a true  iron deficiency  in runners.  Similar evidence  that  iron  supplementation will help prelatent/latent iron deficient athletes is scant (Pate et al., 1979; Vellar and Hermansen, 1971; Celsing et  94 al., 1986; Schoene et al., 1983). The only study to date to measure human muscle enzyme activities when the body iron stores were severely diminished or depleted was that of Celsing et al. (1986). These authors used repeated venesections and reinfusion to induce an artificial latent iron deficiency; they found that this state did not affect endurance,  nor the maximal activities  enzymes in human skeletal muscle. are in  of various  The findings of Celsing et al.  apparent conflict with the previous rat studies of Davies et  al. (1982) and Finch et al. (1976,79).  One possible reason for the  discrepancy could be that extrapolation of findings from weanling rats to adult humans is not accurate.  As well, the rats were  rendered severely iron deficient with a low iron diet while Celsing et al. used  less extreme  experimental conditions  venesections)  with his nine male subjects.  cytochrome oxidase activity, the only  (repeated  Mean values of  iron dependent  enzyme  investigated by Celsing et al. (1986), were slightly lower after retransfusion than in the control state, but this difference did not reach significance (3.6 ±1.7 vs. 4.4 ±1.8 u,mol 0 ( m i n g 2  wet  weight) ). -1  Certain symptoms which occur in other chronic anemias are often observed in iron deficient subjects in the absence of anemia. These nonspecific syptoms include headache, fatigue, heartburn, changes in appetite, vasomotor disturbances, muscular cramping, dyspnea,  palpitation,  menorrhagia  performance (Conrad and Barton, 1981).  and  diminished  work  Most investigators have  95 attributed these functional disturbances to decreases in iron containing enzymes in various body tissues, although a direct cause and effect relationship has been difficult to establish (Conrad and Barton, 1981).  Wood and Elwood (1966) found no  significant difference between treated (iron supplemented) and control (placebo) symptoms.  patients in regards to influence upon these  Repeated measurement of work capacity would have  aided the study of Wood and Elwood (1966).  There may be a  difference between clinical symptoms and physical work capacity as  reference  symptoms.  There  is  higher  precision  in the  determination of physical work capacity.  Ericsson (1970) reported a greater increase in physical work capacity (based on bicycle ergometer heart rate measurements) with  iron treatment (120 mg/day)  in normal elderly  compared to controls of similar age.  subjects  This significant increase  was 4% greater than controls in the men and 12% greater in the women.  Interestingly,  this increase in performance was  matched by an increase in hemoglobin levels.  not  In the placebo  (control) group, a relationship approaching significance (r=.493; 0.01 < p < 0.05) was found between iron storage (stainable iron in the bone marrow) and work capacity.  In other words, 25% of the  variance in work capacity can be explained by the variance in iron storage.  In the control group, physical work capacity increased in  some subjects and decreased in others.  Those control subjects  who initially had greater iron depots were in an advantageous  96 position for improving work capacity.  In the presence of such a  relationship, it was also expected and found (r=0.484;0.01 < p < 0.05) that the amount of stainable bone marrow iron would have decreased in those whose physical work capacity had increased. Depot iron had less influence in the treatment group with high iron intake.  Ericsson (1970) concluded that in apparently  healthy  individuals the increase in physical work capacity during moderate training is related to the availability of iron.  Studies by Ohira et al. (1978, 1979, 1981) indicated that there are beneficial effects of iron treatment that cannot be related to changes in Hb.  Ohira et al. (1981) noted from unpublished data that  even normal rats absorb greater amounts of within four days after oral iron treatment.  5 9  F e in cardiac muscle  Using human subjects  with a wide range of hemoglobin (6.4-14.1 gdl" ) and serum iron 1  levels  (28.5-98  ugdl" ), 1  Ohira et al. (1981) showed that this  effect is more significant in iron deficient groups than in normal iron groups, even when both groups had similar Hb (i.e. 11.8 gdl" ). 1  11.9 vs.  Work capacity was measured before and after one  week of iron treatment (Imferon IV) in the study by Ohira et al. (1981).  There was a tendency for all groups to increase their work  time, although this could merely reflect some degree of adaptation to the treadmill tests.  The post exercise venous blood lactate  data of Ohira et al. (1981) suggests a non Hb related effect. groups of subjects:  Three  high Hb-high serum iron, marginal Hb-high  serum iron and marginal Hb-low serum iron, had almost identical  97 work capacities, thereby eliminating work time as a variable in affecting blood lactate.  The coefficient of correlation (r=0.41; p <  0.05)  iron  between  serum  and lactate levels  after  exercise  suggested that the lower the serum iron, the higher the blood lactate after exercise.  Looking at the post exercise lactate levels  of the two marginal Hb groups (11.9 and 11.8 gdl" ), but one with 1  low serum iron and the other with high serum iron values, significantly higher post exercise blood lactate occurred in the low serum iron group even though exercise were similar.  intensity and duration of  Changes in mean heart rate at a given  heavy workload showed the same relationship.  Schoene et al. (1983) examined the effects of iron therapy on work capacity and exercise induced lactate production in trained, mildly  iron  performance  deficient on  a  female cycle  athletes.  ergometer  Although with  a  exercise  progressive  work-exercise protocol did not change with two weeks of oral iron therapy, lactate production after exercise decreased significantly. Two  weeks of ferrous  sulfate supplementation (300  mg/day)  brought hemoglobin values from an average 12.2 gdl" to 12.7(p < 1  0.05), and ferritin from 10.0 to 22.1 ngml"  1  (p < 0.002).  Blood  lactate levels at maximum exercise in this iron deficient group dropped from 10.3 u,moll" before therapy to 8.42 after therapy (p 1  < 0.03).  The non-iron deficient controls had no significant changes  for any of these parameters.  The results suggested that work  capacity might have improved.  A protocol entailing an endurance  98 test instead of a short, intensive test might have illuminated this possibility  (Schoene  et  al.,  1983).  Continuation  of  iron  supplementation for two months may also have led to a significant improvement in exercise performance as this period of time may be needed for replenishment of iron stores after the hemoglobin concentration  has reached normal, non-anemic levels  1985; Valberg, 1980).  (Jacobs,  The rapid rise in ferritin values noted by  Schoene et al. (1983) probably does not represent increased iron storage  levels,  but  instead  a transient  increase  in  ferritin  synthesis (Jacobs, 1985).  Because evidence concerning the effects of  prelatent/latent  iron deficiency is limited and the complexity of iron usage and depletion is immense, the significance of iron deficiency without anemia is speculative.  Jacobs (1985) states that iron deficiency  anemia, resulting from an impaired iron supply for erythropoiesis, occurs at the same time as the iron supply to other organs is reduced.  Dallman (1978) acknowledges that deficiencies of tissue  iron compounds generally occur in conjunction with anemia, but notes that they may also be observed in its absence.  Clement  (1981) speculates that the non-anemic iron deficient symptoms reported initially  are certain  hemoglobin.  an  indication  enzymes  that  (and  iron  perhaps  deficiency myoglobin)  will and  impair later  99 Appendix B  Definitions WORK CAPACITY: Alactic power - is a measure of explosive power, relying mainly on stored adenosine triphosphate (ATP) and phosphocreatine (PC) in muscle without a significant build up of lactic acid.  On the  Wingate bicycle ergometer test it is the maximal power output observed averaged over a 5 second period and is expressed in watts-kg"  1  body wt.  Lactacid power - is a measure of power output when lactic acid tolerance is maximally stressed. the average watts-kg"  1  On the cycle ergometer test it is  power output over 30 seconds with units being  body wt.  Lactacid  capacity - is the total energy  output during the  anaerobic speed test and is expressed as time to exhaustion in seconds. Anaerobic threshold  (AT)  - is the inflection point of a blood  lactate curve in an incremental exercise test.  It can be expressed  as a %v"0 max or, as in this study, a treadmill speed.  Instead of  blood  exchange  2  lactate  sampling, the  protocol  utilized  gas  variables to indirectly determine the anaerobic threshold and technically threshold.  this  inflection  point  is  called  the  ventilatory  100 V0 max. - indicates the highest oxygen uptake (mlkg" min" ) an 1  2  1  individual can attain during physical work while breathing air at sea level.  On the progressive workload treadmill test this usually  occurs close to the point of exhaustion.  Maximal treadmill velocity - is the highest treadmill velocity (mileshr" ) achieved during the incremental treadmill test. 1  IRON STATUS:  Serum Ferritin:  is a sensitive index of the earliest stage of  iron deficiency-depletion of body iron reserves (Valberg,  1980).  One ng/ml of serum ferritin corresponds to approximately 5-9 mg of storage iron.  Levels below 20 ng-ml"  1  are associated with  absent bone marrow iron (Pakarinen, 1980). ng-ml"  1  Levels up to 64  may still represent an iron deficient state  (Heinrich,  1977).  Hemoglobin (Hb):  is an iron containing conjugated protein found  in red blood cells and capable of transporting oxygen and carbon dioxide to and from the working tissues. Total Iron: the  clinical  (also called plasma iron and serum iron) is useful in evaluation  of  iron  deficient  erythropoiesis.  Exhaustion of the iron stores affects the supply to the plasma pool and hence to the erythroid bone marrow (Bothwell et al.,1979).  101 Unsaturated  Iron  Binding  Capacity  (UIBC):  measure of iron deficient erythropoiesis.  is also a  It is determined by  incubating a known ferrous standard with serum to saturate the available binding sites on serum transferrin. UIBC is the amount of iron that can be taken up by the transferrin.  This figure is added  to the total iron to yield the total iron binding capacity (TIBC).  % Transferrin Saturation:  is a function of both the total iron  and the transferrin concentration.  A saturation of less than 16%  has been shown to be inadequate to meet basal erythropoietic needs  (Bothwell  et  al,  1979).  The  figure  is  arrived  at  mathematically from the values above; total iron/TIBC.  IRON DEFICIENCY: A state which develops  if the loss of iron is greater than the  amount obtained (Pakarinen, 1980). divided into three stages:  (a)  Iron deficiency is commonly  prelatent,  (b)  latent, and  (c)  manifest (Clement and Sawchuk, 1984). a)  Prelatent iron deficiency:  is the earliest stage of iron  deficiency characterized only by a decrease in decreased serum ferritin) (Strauzenberg et al., 1981).  and  storage iron (and  increased intestinal absorption  102  b)  Latent iron deficiency:  prelatent  iron  deficiency,  in addition to the  the  levels  of total  indices of  serum  iron  is  decreased, the unsaturated iron binding capacity is increased, and the % transferrin saturation is decreased (Strauzenberg 1981).  The lack of reliability in these measurements (Pakarinen,  1980) makes differentiation between deficiency difficult.  prelatent and latent iron  For this study, these measurements did not  affect the screening procedures stated  et al.,  statistical analysis.  nor were they utilized in the  Instead,  they  provided  back-up  information on each subject, which may help in the interpretation of the data.  The criterion for prelatent/latent iron deficiency was  serum ferritin levels below 20 n g m l " . 1  Levels below this are  associated with absent bone marrow iron (Pakarinen, 1983; cited in Clement and Sawchuk,1984). c)  Manifest iron deficiency:  iron deficiency characterized (Clement and Sawchuk, 1984). levels below 12 gdl"  1  refers to an advanced stage of by a drop  in hemoglobin  levels  In this study, the criterion will be  for women (Williamson, 1981).  iron deficiency will also be referred to as anemia.  Manifest  103  PHYSICAL ACTIVITY: This criterion for inclusion in the study was defined as regular (three or more times per week) participation in relatively intense (three-quarters  maximal effort) physical activity of at least 120  minutes total duration per week. running.  For most subjects this involved  104 Appendix C  Limitations: SUBJECT SELECTION: The subjects selected were physically active but mostly non-elite, as compliance to the treatment procedures may have optimal training/diet  sacrificed an  regimen.  SAMPLE SIZE: Primarily for practical reasons, the sample size was set at 40 completed subjects.  Calculations using work capacity data from a  similar population supported a size of 40 as being reasonable in obtaining a statistically significant result. USE OF SERUM FERRITIN: A serum ferritin assay allows accurate assessment of prelatent iron  deficiency  but not differentiation  between  prelatent and  latent iron deficiency.  USE OF EIGHT WEEK TREATMENT PERIOD: While this length of treatment was felt necessary to induce a significant repletion of iron stores, the number of dropouts (due to various uncontrollable circumstances) increases with the length of study.  105  PHYSICAL FACTORS: Training, diet, menstrual blood loss and other physical factors which  may affect the testing were  precisely measured.  Instructions  not  rigidly  controlled  or  were given to the subjects  regarding exercise and diet prior to blood tests and work capacity tests.  As well, they were instructed to maintain their typical  training and diet regimen during the treatment period.  A log book  of training, menstrual blood loss, and any other physical factors which may have influenced the testing was completed by each subject.  A three day dietary  analysis prior to both the pre-test  post-test aided in monitoring diet (See Appendix G).  and  106 Appendix D  Delimitations:  This study was delimited to physically active females, age 18 to 40, with prelatent/latent iron deficiency.  Subjects  received  placebo or iron supplementation (320 mg ferrous sulfate = 100 mg elemental iron taken as SLOW-Fe® one tablet BID as tolerated) for a treatment duration of eight weeks.  The  only  blood  parameters  measured were  serum  ferritin,  hemoglobin, total iron, UIBC, and % transferrin saturation. only physical performance measures were  The  the Wingate bicycle  ergometer test, anaerobic speed test, anaerobic threshold, and V 0 max. and max. velocity.  The subjects selected were all  who resided in the greater Vancouver area.  2  volunteers  They underwent a brief  medical examination and history review prior to enrollment in the study, including temperature, stool and urine examination, blood pressure and heart rate determinations.  107 Appendix E THE UNIVERSITY OF BRITISH COLUMBIA FACULTY OF MEDICINE DIVISION OF SPORTS MEDICINE John Owen Pavilion  • Ian Newhouse  Subject Consent Form The purpose of this research is to assess the effects of prelatent/latent iron deficiency in physically active females (see attached abstract). As a subject you will undergo the following tests: (requiring approx. 10 hours). 1. anthropometric tests: underwater weighing for % body fat, height, weight. 2. urinalysis: for hemocyturia (blood loss in urine). 3. stool analysis: for occult blood loss. 4. blood tests: samples will be analysed for serum ferritin, hemoglobin, total iron, unsaturated iron binding capacity, and % transferrin saturation. 5. Wingate anaerobic power test: a 30 second bicycle ergometer test. 6. Anaerobic speed test: a treadmill run (at grade of 20% and speed of 7 mph) to exhaustion (usually between 30 seconds and 1 minute). 7. A progressive workload treadmill test (a treadmill run with velocity increasing every minute up to exhaustion). Continuous sampling of expired gases will allow measurement of VC^max. Testing will be be performed pre and post treatment. All testing will be conducted by skilled technicians.  108 The treatment will be 8 weeks of either iron supplementation (a daily dosage of 320 mg ferrous sulfate (100 mg elemental iron) taken as Slow Fe® twice a day, or matching placebo tablets taken as one tablet twice a day). Placebo subjects will receive iron supplementation upon completion of the post tests. A medical examination will be provided prior to entry into the study. You will be asked to record all training, an estimate of menstrual blood loss, and any other factors which may influence testing for the duration of the testing and treatment period. As well, a 3 day dietary record will be filled out prior to the pre and post tests. Training and diet should be kept consistent with previous habits. Ingestion of anti-inflamatories or medication which may cause acute or chronic blood loss from the upper gastro intestinal tract will not be allowed. The chief agent in this category is acetylsalicylic acid (aspirin). Although you will be exercising to the point of temporary exhaustion, and taking large doses of iron supplementation, there is very little risk involved if you are a healthy, mildly iron deficient individual. The discomfort associated with testing and treatment, apart from temporary exhaustion in the physiological tests, should be mild. At the point of exhaustion on the treadmill tests you will grasp the safety rail and the treadmill will be stopped. If you stumble or fall while running, the treadmill will be stopped and the consequences would be similar to if you had fallen on a track. Blood sampling will be done by skilled technicians. The amount of blood drawn will be small and there will be little discomfort with the procedure. There may be slight bruising at the point of puncture. Side effects of iron supplementation have been reported to affect a small percentage of the population and these include nausea, constipation, and abdominal pain (Gomez & Gomez, 1969). Publication of results will not reveal subject identity as subjectsd will be referenced by number. -2-  109  I have read and understand the above explanation of the purpose and procedures for this test and agree to participate. I also understand that I am free to withdraw my consent at any time.  Signature  Witness  Date  -3-  110 Appendix F THE UNIVERSITY OF BRITISH COLUMBIA FACULTY OF MEDICINE DIVISION OF SPORTS MEDICINE John Owen Pavilion  Ian Newhouse  Subject Consent Form: Muscle Biopsy This project is designed to investigate the response of iron containing skeletal muscle enzymes to iron supplementation. To do this you will be required to have two needle biopsies: one will be taken at the beginning of the study and the second after 8 weeks of iron supplementation. There are minimal risks associated with this procedure. A 1/2cm incision will be made under local anaesthetic in the lateral thigh and a needle inserted in the vastus lateralis. A small portion of muscle (100mg) will be taken with the needle. The location is free from underlying deep structures such as arteries, veins, or nerves. Complications, which are rare, include infection, bleeding, hematoma or an allergic reaction to the local anaesthetic. All procedures will be performed using sterile technique by experienced physicians. Pressurized bandaging will prevent post operative bleeding. There will be some discomfort associated with this procedure at the time of needle insertion but most subjects report this as mild. You will be able to resume your normal activities immediately. All information derived from this project is confidential. In reporting the data, names of the subjects will not be used. Information about your results is available to you and these can be discussed with one of the investigators. We will be happy to answer any questions that you have concerning the project and you are free to withdraw from the project at any time without prejudice to future care. I have read the above consent form and understand fully the procedure and risks involved. I consent to participate in this research project and acknowledge receipt of a copy of this form.  Witness  Subject  Date  111 Appendix G  Dietary Analysis A computerized nutrition analysis was provided by the YMCA. Three days of dietary intake and physical activity were recorded by each subject. The instruction sheets and recording forms are shown on the following pages (pp 112-115).  COMPUTERIZED NUTRITION ANALYSIS: Personal Form  YMCA This evaluation is only as accurate as the information you provide on these forms. To obtain the best results, follow the instructions carefully and complete ALL sections as accurately as possible. Complete: 1. ONE green personal form only. 2. ONE white computer form FOR EACH DAY to be analyzed. SECTION A: PERSONAL DATA No. OF DAYS TO BE ANALYZED.  1 10 7  GROUP.  NAME ADDRESS PHONE NUMBER SEX: MALE •  FEMALE  • .inches OR.  HEIGHT (without shoes)  lbs. OR _  WEIGHT (without clothes) FRAME SIZE: SmallD BIRTH DATE  day  MediumD month  DO YOU WISH TO LOSE WEIGHT?  YESD  NOD  ARE YOU ON A SPECIAL DIET?  YESD.  NOD  VITAMIN/MINERAL SUPPLEMENTS?  YESD  NOD  ARE YOU PREGNANT?  YESD  NOD  AREY0U NURSING?  YESD  NOD  ARE YOU TAKING  .cm  -kg  LargeD  year  SECTION B: FOOD RECORD t.  Use a worksheet (see sample) to list EVERYTHING  TIME OF DAY?  YOUftTEOR DRANK for EACH DAY to be analyzed. Be sure to include:  a)  ALL FOOD AND DRINKS, for example: snacks, sugar and cream in coffee, mayonnaise in a sandwich, sauce on  10-16 ...  HOW MUCH?  FOOD OR DRINK?  coffee.  am  IT-CO r\oov\  vegetables, candies, soft drinks, wine, etc. •  b) THE TIME OF DAY these foods were consumed. c)  2.  Ji  l  "U»p • .  *  THE AMOUNT OF FOOD that you ate (ounces, slices, cups, teaspoons, etc., in whole or decimal numbers).  On the WHITE COMPUTER SHEET, enter the NUMBER OF SERVINGS of ALL foods and drinks ACCORDING TO LISTED SERVING SIZE. a) Break down all combination foods. (For example, cheese omelette in food record above.) c) WRITE the number of servings you had in the CORRECT TIME SLOT  b) NOTE THE SERVING SIZE  FOODS EATEN  ft  N 0  Serving S i z e ^ . ^5 am #  11 anrlL  Q^RVING^ 11 am -  5 am  CODE Office  Use Only  MEAT, FISH, EGG OR POULTRY  EGG Cheese: Cheddar Vegetable Oil  1 1 oz. 1 tbsp.  2. I.S as  10129 10009 40518  Program originally developed by Action B.C. Copyrighted 1983. This form may be reproduced with the consent of the Vancouver YMCA, 955 Burrard Street, Vancouver, B.C. V6Z1Y2 (604)681-0221  TIME OF DAY  LENGTH OF TIME  ACTIVITY  LEVEL*  z  SECTION C: ACTIVITY RECORD 1.  Use a worksheet (see sample) to RECORD YOUR ACTIVITY FOR EACH DAY, (that is, tor 24 HOURS.)  2.  2.  '/2 I A X .  Activity levels explained below.  0.-O5-0M5  TOTAL THE NUMBER OF HOURS SPENT AT EACH LEVEL. The total must EQUAL 24 hours.  /CP 5  3 /  3  wi/H.  4 rvK  office  wov'l*  2  WRITE THE TOTAL HOURS spent at EACH LEVEL in the ACTIVITY section at the top of the WHITE  COMPUTER FORM. ACTIVITY: Hours at each level  1  e.s  Sleeping/Resting  ?  \2-0  Sitting/Standing  ,  9.0 Light Activity  4  0.5  Active  5.  0 Very Active  = 24.0 Hours  •EXAMPLES OF LEVELS OF ACTIVITY  LEVEL 2  LEVEL 1  Sitting, eating, watching TV. classwork, office work, driving, playing quietly, playing a musical instrument, sailing, STANDING, personal toilet, teaching, laboratory work, HOUSEWORK, SHOPPING, cooking, bowling, pool  SLEEPING, resting  LEVEL 3 (Count only the TIME that you are  ACTIVELY PARTICIPATING) BRISK WALKING (2-3m.p.h.), BICYCLING (5.5m.p.h.), farming, gardening, mechanical work, electrical trades, house-painting, restaurant trades, loading/stacking, carpentry, dancing, 4 baseball, weight training, golf, l^y-W^-i RECREATIONAL swimming, table tennis tennis, volleyball, canoeing or rowing  LEVELS 4 and 5: Activities at these levels should cause you to raise  *  your HEART RATE, to BREATHE HARD, to PERSPIRE, to use ALL YOUR EFFORT. Count only the TIME that you are  ACTIVELY PARTICIPATING.  LEVEL 4 WALKING/JOGGING (4.5-5m.p.h.), hiking, BICYCLING (10m.p.h.), digging, skipping, downhill skiing, recreational basketball, badminton or skating, canoeing (4m.p.h.), competitive table tennis or tennis  /  LEVEL 5 RUNNING (more than 5m.p.h), football, hockey, soccer, lacrosse, bicyling (13m.p.h.), COMPETITIVE badminton, volley ball or basketball, racquet sports, vigorous skating or rowing, cross-country skiing, martial arts, swimming (45 yards/min. or more  CHECK THAT YOU HAVE COMPLETED ALL SECTIONS OF THE GREEN PERSONAL FORM AND WHITE COMPUTER FORM(S) BEFORE RETURNING THEM.  COMPUTERIZED NUTRITION ANALYSIS: Computer Form GROUP  NAME. DAT.E_  Day  Month  A C T I V I T Y : Hours at each level  1.  Sleeping/Resting  Sitting/Standing  NO  FOODS EA1EN  Serving Size  E V A L U A T I O N DAY N U M B E R .  Year  OF S E R V I N G S  5 am • It a m  11 a m 5 pm  5 pm • 5 am  CODE Ollice U s e Only  MILK AND MILK PRODUCTS  Light A c t i v i t y  1 to 7  .5._  Active  NO  F O O D S EAIF.N  Serving Size  F R E S H : apple  1 med.  24.0 H o u r s  Very A c t i v e  5 am 11 a m  OF SEItVI N G S 11 a m • 5 pm  5 pm 5 am  CODE Ollice U s e Only 13  1 07.  10007  n p i i c o t s ( 3 ) . m a n g o ('/<)  1 seiv.  30  Cheddar, hard  1 oz  10009  banana  1 med.  141  cottage c h e e s e  V? c u p  10015  perries: blackberries, blueberries, raspberries  Vi c u p  1849  1 oz  10017  C h e n ics  1 cup  663  1 serv.  10042  giapes  1 cup  1085  Vi  1053  Vi  1358  CHEESE: camembert, soil  cream cheese p t o c e s s e d (1 s l i c e ) , s p t e a d (2 t b s p . )  1 oz  10040  grapelruit  Ice c r e a m  Vi c u p  10061  m e l o n s : c a n t a l o u p e (orange)  Ice m i l k  Vi c u p  10164  1 cup  4046  S w i s s or g o u d a  Instant b r e a k f a s t : w i t h m i l k M I L K : chocolate  h o n e y d e w (pale green) watermelon  2 " wedge  1360  10"x1" slice  2424  1 cup  10103  o r a n g e , tangerine  lined,  1420  condensed, sweetened, canned  1 tbsp.  10095  p e a c h , n e c t a r i n e ('/:), p a p a y a (V?_cyd)  1 serv.  1479  evaporated, whole, canned  1  1502  V i " slice  1611  _  t tbsp.  11096  pear  s k i m , buttermilk  1 cup  10085  pineapple  ?."/.  l cup  10079  plums. prunes  whole, homogenized  1 cup  10077  stiawbemes  Milkshake  10 oz  10111  Y O G U R T : fruit flavoured  6 oz  10122  dates  2  952  plain  6 oz  10116  prunes  2  1818  raisins  B R E A D S : European, d a r k ' truil b r e a d , r a i s i n ' w h i l e , enriched whole w h e a t , w h o l e grain B U N S , R O L L S : plain, hamburger w h o l e wheat C E R E A L S : c o o k e d , w h o l e grain  'A")  '/< c u p  1846  Vi c u p  27  456  grape  Vi c u p  1088  1 slice  452  grapefruit  Vi c u p  1071  t slice  461  orange  Vi c u p  1437  1 slice  471  pineapple  Vi c u p  1619  1  1902  prune  '/; CUp  1821  1  1906  VEGETABLES  Vi c u p  1391  COOKED: asparagus  4 sprs.  47  4005  b e a n s , g r e e n , yellow  Vi c u p  183  Vi c u p  385  1 cup  484  410  1 slice  JUICES (unsweetened):  dry, l l a k e d , e n r i c h e d dry, s h r e d d e d , whole g r a i n  1 cup  4033  beets  dry, s u g a r c o a t e d  1 cup  4037  broccoli  Vi c u p  4101  C R A C K E R S : plain m e l b a toast w h o l e grain M U F F I N S : fruit whole grain, bran PASTA: m a c a r o n i , n o o d l e s , spaghetti P A N C A K E S . W A F F L E S : 4 " diam.  39  apple  1  1 cup  granola  1641 2217  2 halves  DRIED: apricots  BREADS AND CEREALS B I S C U I T S : b a k i n g p o w d e r , s c o n e (Z " x i  1 Vi c u p  4  916  2  4063  Biussels spiouls  8  490  cabbage  Vi c u p  513  carrots  Vi c u p  620  cauliflower  1 cup  631  1 cup  520  2  919  1 med.  1345  Chinese g r e e n s , bok choy  1 med.  1346  c o r n , k e r n e l i'/i c u p ) , cob (1 s m . )  1 serv.  857  1 cup  1300  eggplant  Vi c u p  987  1  1457  m i x e d vegetables  Vi c u p  2404  1598  onions  Vi c u p  1413  P I E C R U S T : plain, l o w e r c r u s l  wedge  RICE:  Vi c u p  1870  parsnips  Vi c u p  1474  Vi c u p  1874  peas  Vi c u p  1530  1878  potatoes: b a k e d , b o i l e d , m a s h e d  1 med.  1788  1 cup  1806  brown white, converted, parboiled white, unenriched  Vi c u p  M I S C E L L A N E O U S : bran  '/< c u p  wheat germ  1 tbsp.  yeast, b r e w e r ' s  1 tbsp.  ..  !  2446  trench d i e s . Iried potatoes  244 7  scalloped  Vi c u p  1795  2478  sweet, y a m  1 med.  2249  spinach  Vi c u p  2170  s q u a s h : w i n t e i . yellow  Vi c u p  2200  FRUITS C A N N E D : (sweetened):  applesauce  Vi c u p  apricots  V: c u p  35  cup  671  fruit c o c k t a i l  V: c u p  1023  peaches  77 c u p  Ml!.'!  pear s  Vi c u p  150?  plums, prunes  Vi c u p  IB.-. 5  rhubarb  Vi c u p  1866  cherries  others - choose F R E S H + 3 t s p . sugar p e i s e r v i n g  summer, zucchini  HAW  Vi c u p  2192  tomatoes, b a k e d , c a n n e d  Vi c u p  2284  turnip:'.  Vi c u p  2353  Vi c u p  803  p i t i e d vegetable  '/) c u p  4059  pnfalu. w i t h d r e s s i n g  Vi c u p  1812  s. l.;«.h  ri?. ^ " ''' ,  w,  avncarln canols  ,  ;,vn,,,,!,Ko  1T'  64  1 med.  619  CoDvrinht 1 9 8 3 . This form m a v be r e p r o d u c e d with the consent of tho  releiv  1 stalk  637  Vancouver. Y M C A . 9 5 5 B u r r a r d Street. Vancouver, 3 C. V 6 Z 1Y2  C'.icumbei  7 slices  943  (604)  g i e e n n e p p e i . du.j:d  '/< c u p  1545  681-0221.  NO FOODS EATEN  OF S E R V I N G S  Serving Size  5 am tl am  11 a m 5 pm  5 pm • 5 am  CODE Ollice U s e Only  NO . OF S E R V I N G S F O O D S EAI E N  Serving Size  lettuce, c h o p p e d  1  cup  1258  DESSERTS, SWEETS, BAKED GOODS  mushiooms  / ' < cup  1351  Jam.  onions, green radishes sprouts  bean  1  allalla  tomato J U I C E S : tomato, vegetable  1  t  2  T  jelly, hnney. s y m p  -115  Molasses  8-1-1  Sugar: white, biown  'A r . u p  1IM  S w - e i sauce  1 med.  2282  'h c u p  2288  C A N U V : hard(fi) c a r a n i e l s ( 3 )  MEAT. FISH, EGG OR POULTRY EGG  1  F I S H : c o d . halibut, sole: b a k e d  6 oz  listi s l i c k s  1  t r e s h w a t e r fish  3oz  salmon, tuna: baked  3oz  canned S H E L L F I S H : clams(5).  11)1?'.!  3oz  d e e p Iried  cup  oysters(9)  1  limping  1Ibsp.  1148  1ibsp.  1341  1 tsp.  2230  2 Ibsp  761  1 serv.  608  1 piece  680  chocolate bar  1  587  chocolates  1  C A K E : a n g e l lood  100  Inn!  594  2vr  521  wedge 2"x " ' IW'x'/V  532  plain, n i l icing  2'/r-" s q .  534  •1089  t i c l i . with icing  2'/;"  526  1958  COOKIES: brownies, squares  1 (11  2324  sq.  1  813  chocolate chip  1  818  773  gianola bats  2  4101  3oz  905  oatmeal  1  4069  1  812  1  958  shrimp, scallops  3oz  20-15  M E A T : b e e l , v e a l : g r o u n d , h a m b u r g e r patlie  3oz  370  Doughnut. D a n i s h pastry, eclair  roast  3oz  2-187  Fiuil crisp  steak  6 oz  310-1  J e l l i e d d e s s e i l . jello  s t e w meat  3oz  353  d e l i - s t y l e m e a t s (1 s l i c e )  1  l a m b : roast, c h o p liver, o r g a n m e a l s  oz  Fie (3'/,-'  Vt c u p  3oz  1230  PUDDING, e g g c u s t a i d  3 oz  12G7  1 oz  19815  1  oz  2006  2 slices  126  948  rice, fapioca  Vi c u p  1891  Vt c u p  10066  Sherbet BEVERAGES OR DRINKS  12  lioueur(1 o z ) , d e s s e r t wine( 1V? o z )  1  2014  liquor  6 ribs  1762  t  1999  Iried t u r k e y : roast  1144  1826  3oz  P O U L T R Y : c h i c k e n ; roast  1  Vt c u p  1717  weiner (frankfurter)  1566  Vi c u p  1769  roast, c h o p s (2 s m a l l )  1032  1  milk  3oz  s a u s a g e s ( 4 " link)  25  Vi c u p  w e d g e ) , far!, turnover  P o p s i c l e . ices  liver pate  ham, hack bacon  other, n s s o i l e i l  1982  l u n c h e o n meat, c a n n e d  spareribs  CODE Ollice U s e Only  5 pm 5 am  I serv.  c r a b , lobster  p o r k : b a c o n (side)  11 a m 5 pm  chewing gum  795  /  rnarshmnllows(4  5 am 11 a m  A I X U M t l l : beer  1  400  1 Vi oz  398  w i n e , lalile Carbonated  pop m o i i - d i e l )  394  oz  4 oz  401  10oz  404  3oz  50019  Colfee  1 cup  4051  3 oz  50008  F r u i l flavourer! d r i n k ( c r y s t a l s )  1  4043  3 oz  50166  Hot chocolate (no m i l k )  1 cup  779  Lemonade  1 cup  1252  MEAT ALTERNATES  cup  Vt c u p  160  P o s t u n i , coffee s u b s t i l u l e s  1 cup  4070  lima  Vt c u p  177  lea. (il i c e d , a d d 2 Isp. s u g a r )  1  4050  soy  Vt c u p  2140  while  Vi c u p  155  B E A N S , C O O K E D : kidney, r e d  cup  SNACKS Corn c h i p s 01 s n a c k s , c h e e z i e s  1 sm. pkg.  1  4080  Lentils  '/? c u p  125-1  P o p c o r n ( w i l h oil a n d sail)  Split peas  Vt  1533  Potato c h i p s  1 sm pkg.  1809  Vt c u p  1-196  Pretzels  1 sm. pkg.  1814  V. c u p  1-195  COMBINATION DINNERS  N U T S : salted, roasted unsalted, raw  Cup  cup  1655  Peanut butter  2Ibsp.  1-199  B a k e d b e a n s w i l h tomato s a u c e  1  cup  156  Seeds: sunflower, sesame  2 tbsp.  2236  Chili w i l h beans  1  cup  756  2oz  21-15  C h o p si.iey w i t h m e a l  1  cup  Macaroni and cheese  1 cup  1304  3 oz  2007  Tofu: b e a n c u r d SOUPS, SAUCES, CONDIMENTS, OTHER S O U P S : b r o t h : c o n s o m m e , plain  1 cup  2065  M e a l loal  cup  2I08  M e a l pie (1 s m a l l . 3 W  t cup  2067  P i z z a w i t h c h e e s e , tomato s a u c e  c h o w d e r , c l a m (no m i l k )  t cup  2085  S p a g h e t t i w i t h c h e e s e , tomato s a u c e  1 cup  c r e a m s o u p s , all (with m i l k )  t cup  2090  S l e w m e a l with v e g e t a b l e s  1  t cup  2097  FATS, CREAMS AND OILS  %  cup  -108-1  Duller. 1 p a l  tomato  Vi cup  229G  C R E A M : coffee, t p k g .  w h i l e , (if c h e e s e , a d d V? oz C h e d d a r )  V* c u p  2-170  SHIM  1:17:1  •.-.•iiilUi-'d  / ' < cup  10053  with vegetables  1  with n o o d l e s  lentil, split pea S A U C E S : gravy  Mustard P i c k l e s (2 s l i c e s ) , r e l i s h (1 t h s p . ) Soy  762  1 IS|, 1  1  wedge) 6"  382  wedge  1628 2163  cup  371  1 isp.  10001  1 tbsp.  10049  1 Ibsp.  10056  1 •.;,'>  Coltfi.! .-.iril'm-.-r. i n . - a r n -.ubslilufe  1  tsp.  10069  ? 1 Mi  Margarine  1  isp.  43130  sauce  I Ibsp.  tomato k e t c h u p  1 Ibsp.  V ' m e l n h l ' - ml  1 Ibsp.  40518  Lemon juice  1 Ibsp  S A L A D D R E S S I N G S , blue c h e e s e  1 Ibsp.  45539  b e n c h , oil a n d v i n e g a r  1Ibsp.  45120  low 1,-11. d i e l  1 Ibsp.  45023  mayonnaise  1Ibsp.  45018  1 h o u s a u d Island  1 Ibsp.  I  45017  116 Appendix H  Schedules of examinations and tests  Visit # Week Pretreatment  Evaluation  1  2  3  4  -1  0  4  8  X  3 Day Dietary Analysis  X  X  Blood Loss Studies  X  X  Hematological Tests  X  X  Patient Consent  X  Physician Examination  X  Randomization  X  Anthropometric  Tests  X  X  Physiological Tests  X  X  Dispense Study Medication  X  X  Collect Unused Medication Dispense Patient Diary*  X X  Collect Patient Diary Study Termination Form  * Patient diary monitored menstrual blood loss, training including type, duration and intensity, use of concomitant medications and study medication.  X X  X X  117  Appendix I  Iron Status Raw Data #  Ferritin  Hemoglobin  N o r m s : : 10-160 na/ml Pre Post  12-16 a/dl Pre Post  Iron Total 41-130 iia/dl Pre Post  U.I.B.C. 105-282 ua/dl Pre Post  % Saturation 20-55 % Pre Post  Iron Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18  8 7 10 18 5 10 8 7 18 14 18 19 11 11 19 14 10 16  63 35 14 83 19 29 30 12 26 33 34 40 44 37 27 29 62 75  12  12  24  Mean  12.4 37.7  13.5 12.9 13.8 12.9 12.5 13.9 12.6 13.1 13.1 12.9 12.9 14.4 14.7 13.2 12.9 13.7 13.7 13.3 13.9 13.4  13.6 12.6 13.1 13.2 13.3 14.5 13.4 14.1 13.1 12.9 13.3 13.6 14.3 13.5 13.4 13.2 13.4 13.8 13.4 13.5  14.5 13.1 13.0 13.0 13.4 13.4 12.2 12.1 12.3 12.6 13.1 12.7 13.7 13.4 13.5 12.6 12.2 12.7 12.2 12.2 12.7 13.0  13.7 13.8 12.9 12.8 14.3 13.5 12.2 13.4 12.7 13.1 12.8 12.7 12.7 13.6 12.9 12.8 12.9 13.6 10.9 11.1 11,2 13.1  166 118 87 107 95 86 114 33 67 53 60 196 47 45 87 103 39 70 66 76 144 69 221 60 71 66 124 48. 79.8 98.5  66 130 63 60  384 209 199 348  278 243 291 309 286 214 208 250  54  251.8  158 295 210 164 219 387 197 250 296 260 143 221 235 202 222 96 240 194 103 195.7  15 38 24 15  51 29 29 39 30 18 37 12 18 17 58 17 27 26 26 60 20 27  18 16 26 11 19 24 52 21 47 55 25.1 34.1  Placebo Group 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39  15 19 10 8 10 10 15 8 7 17 14 18 8 15 19 11 7 9 13 5  4Q  1Q  Mean  12.2  23 25 8 12 27 17 14 5 6 19 12 21 40 20 15 26 12 8 13 7  5  17.2  69 85 55 137 179  68 158 44 74 59 102 133 54 27 94 45 81 60  84 81 54 16 122 95 112 50 38 116 29  25 160 91 28 155 30  as  52  84.1  70.5  230 307 294 225 164  209 274 246 387 153 269 220 312 243 292 389 272 252.4  198 205 253 263 231 233 330 385 385 277 257 224 276  23 22 16 38 52  26 44 15 22 20 30 29 12 7 25 14 27 18  282 181 263 336 241 382  29 24 18 4 44 26 34 14 14 28 7  2ZZ  22 13.  231.9  8 47 26 8 39 7  25.6 21.4  118 Appendix J  Work Capacity Raw Data # Alactic P. Pre  Lactacid  A.S.T  VOoimax  AT-  Max., VeL  Post  Pre  Post  Pre  Post  Pre  Pre  Post  1 7.7 7.2 2 7.1 7.9 3 8.0 9.1 4 9.3 9.0 5 8.8 9.8 6 7.6 7.5 7 8.1 8.8 8 8.9 8.2 9 8.6 8.1 10 9.4 7.8 11 8.8 7.9 12 9.7 9.6 13 10.1 7.3 14 8.8 8.8 15 10.0 7.3 16 7.7 6.1 17 8.0 9.7 18 10.8 10.0 ia 2A Mean 8.8 8.4  6.4 5.0 6.4 6.9 7.3 6.3 6.6 8.0 6.4 7.4 6.9 7.2 7.1 6.9 7.9 5.6 6.5 8.9  29.0 26.0 21.0 71.0 36.0 47.5 39.0 51.0 42.0 35.0 40.0 38.0 33.0 31.5 38.0 29.0 34.5 63.5 41.3  29.0 25.0 28.5 94.5 45.0 48.0 46.0 59.5 40.5 47.0 44.0 38.0 33.5 37.0 39.5 29.5 41.5 67.5 74.0 45.1  6.5 8.5 8.0 9.5 8.0 9.0 6.0 7.0 7.0 7.5 6.0 7.0 7.0 7.5 7.0 6.5 6.5 8.0  6.9  6.4 6.0 7.3 8.0 8.2 5.9 7.0 7.5 6.6 6.3 6.3 7.7 6.6 6.6 6.7 5.2 7.3 8.0 IA 6.9  6.5 8.0 7.5 9.5 8.0 9.0 6.5 7.0 7.5 7.5 6.0 7.5 6.5 7.5 7.5 6.5 7.5 8.0 M 7.4 7.5  45.2 43.5 47.8 63.5 51.1 58.5 50.6 53.8 47.3 54.0 51.7 48.6 51.8 52.4 45.0 43.4 48.2 60.9 57.3 51.3  45.4 46.7 57.8 54.7 56.3 52.8 52.3 55.0 54.8 56.0 49.7 51.6 50.2 53.6 46.5 51.0 53.7 59.7 53.2 52.7  9.0 8.5 9.5 9.5 9.0 9.0 12.0 12.0 10.5 10.0 11.0 11.0 9.5 9.5 9.0 10.0 10.0 9.5 10.0 10.0 8.5 9.0 10.0 9.5 8.5 9.0 9.5 10.0 9.0 9.0 9.0 9.5 9.5 9.5 11.0 10.5 11.0 10.5 9.8 9.5  6.2 7.1 5.8 7.1 6.6 6.9 7.8 7.2 7.3 8.6 7.8 6.2 7.4 6.7 6.7 6.6 8.0 6.7 5.6 6.4  6.5 6.7 5.6 7.4 6.2 6.5 7.0 6.1 6.9 8.8 7.2 6.9 6.3 7.5 7.8 5.9 8.6 6.1 5.7 7.3  33.0 54.0 29.0 66.0 29.0 35.0 51.5 37.5 45.5 58.0 50.0 36.5 38.0 50.0 45.0 26.5 73.0 28.0 40.5 42.0  34.5 57.5 31.5 66.0 31.0 44.5 60.0 47.5 48.0 65.0 47.5 33.0 39.5 39.0 48.0 26.0 60.0 28.0 31.0 38.5  M  ZZ  50.0 56.6 47.3 54.9 51.4 49.5 56.2 51.1 50.0 53.0 53.0 50.9 43.9 42.0 49.4 39.7 57.4 55.6 49.1 45.1  58,5  55,0  7.0 7.5 7.0 8.5 7.5 7.5 7.5 6.0 6.5 7.5 7.0 8.0 7.0 6.0 8.0 6.0 8.0 7.5 7.5 7.0 IA 7.2  55.5 59.3 50.3 57.2 52.9 47.0 56.0 52.0 47.6 55.6 49.7 48.8 48.1 39.1 51.8 38.8 52.2 48.8 47.7 45.9 55,4 50.6  9.5 9.5 9.5 10.5 9.0 10.0 10.0 8.5 9.0 10.0 9.0 10.0 9.0 8.5 10.0 9.0 11.0 10.0 10.0 10.0 10.0 9.6  &2  79-0  Placebo Group 20 7.6 8.3 21 8.7 7.5 22 6.4 6.3 23 8.8 9.6 24 7.8 7.6 25 9.0 8.0 26 7.6 7.5 27 7.6 6.7 28 8.4 7.5 29 10.0 10.0 30 9.2 8.5 31 6.9 7.8 32 10.4 7.0 33 8.4 8.6 34 7.4 8.2 35 8.4 8.5 36 9.1 12.2 37 8.9 6.9 6.7 38 5.9 39 6.7 8.9 9J2 42 IA Mean 8.4 8.2  7.0  6.0  43.7  44.8  Post  7.0 7.5 7.0 8.5 7.0 7.0 8.0 6.5 6.5 8.0 6.5 8.0 7.0 6.0 8.0 6.0 7.5 7.5 7.0 7.0 IA 7.2  56,5 50.6  Pre  Post  9.5 9.0 9.5 10.5 9.0 9.0 10.5 9.0 9.5 10.5 9.0 10.0 9.0 8.5 10.0 8.0 10.5 10.0 10.0 8.5 10.0 9.5  119 Appendix K Enzyme Activity Raw Data (units g  Subject # Iron Group 34 8 12 19 21 24 31 36 37 as Mean+S.D. Placebo Group 11 20 22 23 35 Mean ±S.D.  Citrate synthase Pre-test Post-test  1  protein) a-GPDH Pre-test Post-test  0.052 0.043 0.037 0.057 0.065 0.048 0.039 0.041 0.033 0,059 0.047 ±.011  0.031 0.048 0.050 0.041 0.052 0.043 0.055 0.040 0.057 0,065 0.048 ±.010  0.038 0.064 0.061 0.086 0.059 0.073 0.045 0.050 0.059 0,12Q 0.066 ±.024  0.135 0.078 0.066 0.082 0.083 0.056 0.098 0.078 0.087 0,087 0.085 ±.021  0.054 0.059 0.021 0.036 0.025 0.039 ±.017  0.056 0.047 0.046 0.036 0.026 0.042 ±.011  0.061 0.087 0.021 0.084 0.036 0.058 ±.029  0.079 0.060 0.070 0.072 0.051 0.066 ±.011  1 unit = 1 umole of product produced under saturating conditions in 1 minute at 25°C.  120 Appendix L  Descriptive statistics of placebo treated subjects who became anemic (n = 3)  Variate  Count  Pre-Test  Post-Test  Serum ferritin (ngml" )  3  9.3±4.0  8.3±4.2  Hemoglobin (gd" )  3  12.4±0.3  11.1±0.2  Iron Total  3  75.0±43.7  81.3±65.4  UIBC (^gdr )  3  317.7±62.6  300.0±73.3  % Transferrin Saturation (%)  3  19.3±11.0  21.3±16.3  Alactic Power (watts-kg" body wt)  3  7.1±0.7  7.9± 1.8  Lactacid Power (wattskg" body wt)  3  6.1±0.5  6.9±1.1  Anaerobic Speed Test (seconds)  3  47.0±10.0  41.5±12.3  Anaerobic Threshold (mh" )  3  7.3±0.3  7.0±0.0  V0 max  3  50.2±5.8  49.7±5.0  3  10.0±0.00  1  1  (wrdr ) 1  1  1  1  1  2  (mlkg" min' ) 1  Max Velocity (mh" ) 1  1  9.5±0.9  


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