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Iron status among infants 8-26 months of age in Vancouver and socio-cultural/dietary predictors of risk… Williams, Patricia Lynn 2001

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Iron status among infants 8-26 months of age in Vancouver and socio-cultural /dietary predictors of risk for iron deficiency anemia By P A T R I C I A L Y N N W I L L I A M S B . S c . H . E c . Mount Saint Vincent University, 1989 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Individual Interdisciplinary Graduate Studies[Health Care and Epidemiology / Health Promotion / Human Nutrition / Pediatrics]) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A 2001 © Patricia Lynn Williams, 2001 UBC Special Collections - Thesis Authorisation Form http://www.library.ubc.ca/spcoll/thesauth.html In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n permission. The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada 1 of 1 3/23/01 4:24 P M Abstract Abstract The feeding practices of Chinese and Caucasian infants may place them at risk for IDA and its deleterious consequences. It is currently recommended that dietary assessment is used to screen 'high risk' infants for risk of IDA however, dietary instruments to assess iron nutrition among Caucasian and Chinese infants are not available. The purpose of this study was to develop and assess the utility of dietary instruments for identifying Caucasian and Chinese infants ages 8-26 mths with poor iron status. Letters describing the study were sent to 1585 parents of potentially eligible infants identified through birth lists and 613 of these parents were contacted by telephone. Of these, 148 infants 8-26 mths of age, n=84 Caucasian, n=48 Chinese completed the study. Capillary blood samples were collected and analyzed for hemoglobin (Hgb), serum ferritin and soluble transferrin receptors (sTfR). A 191-item food frequency questionnaire (FFQ) was developed to provide a comprehensive assessment of the dietary intakes and sources of energy, iron and other dietary factors influencing iron absorption. Feeding history and current diet were assessed using a Socio-Cultural and Infant Feeding Questionnaire, a 3-day food record (3d-FR) and the interviewer-administered FFQ. The 3d-FR and FFQ were analyzed for dietary intakes and sources of energy, iron (total, heme and non-heme), vitamin C, calcium and dietary fibre using Food Processor®. The FFQ measures of total and heme iron intakes showed criterion validity compared with sTfRrferritin ratio (i=-0.33 and -0.27, respectively, PO.001), and relative validity compared with 3d-FR measures of total and heme iron intakes (r=0.65 and 0.72, respectively, PO.OOl). The prevalence of IDA (Hgb <110 g/L + serum ferritin <12 ug/L) was higher at ages 8-12 than 13-26 mths in Caucasian (15% vs. 4%) and Chinese (6% vs. 0%) infants (P=0.001). Low iron stores (serum ferritin £12 ug/L without IDA) was found in 30% of Caucasian and 19% of Chinese infants. The types and quantities of complementary foods fed, most notably the introduction of meats later than 9 mths of age, and subsequent low intakes of meats, in a predominandy breast milk diet were associated with the high prevalence of poor iron status among Caucasian infants. Four key dietary patterns were associated with poor iron status: 1) a history of no iron-fortified formula or supplemental iron; 2) cows' milk fed prior to 9 mths of age; 3) >800 g/day cows' milk/milk products; and 4) <30 g/day meats. Primary prevention initiatives should be targeted to 8-12 mth old Caucasian infants and include ways to ensure adequate intakes of heme iron or alternatives to this, and avoidance of early introduction or excessive quantities of cows' milk. Brief dietary-screening tools for detection of infants at risk for IDA are presented but need to be field-tested in future research. ii Table of Contents TABLE OF CONTENTS PAGE ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES viii LIST OF FIGURES x GLOSSARY OF TERMS xii ACKNOWLEDGEMENTS xv DEDICATION xvii CHAPTER 1. INTRODUCTION 1.1 Background 1 1.2 Purpose of Study 5 1.3 Study Objectives 6 1.4 Study Hypotheses 8 CHAPTER 2. LITERATURE REVIEW 2.1 Introduction 10 2.2 The Importance of Iron in Human Nutrition 11 2.3 Iron Compounds in the Body: Chemical Properties, Distribution and Metabolic Function 14 2.3.1 Functional Iron Compounds 15 2.3.2 Iron Transport Proteins 16 2.3.3 Iron Storage Proteins 17 2.4 Laboratory Assessment of Iron Status among Infants 17 2.4.1 Hemoglobin and Red Cell Indices 20 2.4.2 Erythrocyte Protoporphyrin 21 2.4.3 Transferrin Receptor 21 2.4.4 Serum Iron/TIBC and Transferrin Saturation 23 2.4.5 Tissue Concentrations 23 2.4.6 Quantitative Phlebotomy 23 2.4.7 Serum Ferritin 23 2.5 Iron Homeostasis and Iron Balance durmg Infancy 25 2.5.1 Iron Endowment at Birth 27 2.5.2 Growth Rate 27 2.5.3 Iron Losses 28 2.5.4 Iron Absorption 28 2.6 Recommendations for Iron Intake during Infancy 31 2.7 Sources, Amount and Bioavailability of Dietary Iron in Infancy 34 2.7.1 Human Milk 35 2.7.2 Infant Formulas and Cows' Milk 36 2.7.3 Complementary Foods 37 2.8 Adequacy of Iron Intakes in Infancy 39 2.9 Prevalence of Iron Deficiency in Relation to Risk Factors 45 2.9.1 Infant Age as a Predictor of Risk for Iron Deficiency 47 2.9.2 Ethnic Background as a Predictor of Risk for Iron Deficiency 48 2.9.3 Socio-economic Status as a Predictor of Risk for Iron Deficiency 53 2.9.4 Primary Milk Feeding as a Predictor of Risk for Iron Deficiency 53 2.9.5 Intake of Complementary Foods as a Predictor of Risk for Iron Deficiency .... 54 iii Table of Contents 2.10 Strategies for Prevention and Detection of Iron Deficiency in Infancy 56 2.10.1 Strategies for Primary Prevention of Iron Deficiency Anemia 56 2.10.2 Strategies for Secondary Prevention of Iron Deficiency Anemia 57 2.11 Research Instruments for Assessing Dietary Intakes in Infancy 59 CHAPTER 3. DESIGN AND METHODS 3.1 Study Design and Ethical Approval 63 3.2 Development and Use of Dietary Assessment Instruments 63 3.2.1 Food Frequency Questiomiaire (FFQ) v 63 3.2.2 3-day Food Record (3d-FR) Package 65 3.2.3 Food Composition Database 65 3.2.4 Socio-Cultural and Infant Feeding Questionnaire 66 3.2.5 Validating the Dietary Assessment Instruments 67 3.3 Subjects 68 3.3.1 Participant Identification and Selection Criteria 68 3.3.2 Participant Recruitment 69 3.3.3 Clinic Scheduling 70 3.4 Data Collection 70 3.4.1 Socio-Cultural and Dietary Data 70 3.4.2 Anthropometric Measures 71 3.4.2.1 Body Weight 71 3.4.2.2 Length 72 3.4.2.3 Head Circumference 72 3.4.3 Blood Collection 72 3.5 Data Analysis 72 3.5.1 Pilot Study 72 3.5.2 Sotio-(^tuial and Infant FeedmgG^ 73 3.5.3 3-day Food Record and Food Frequency Questionnaire 73 3.5.4 Hematological and Biochemical Analysis 75 3.5.4.1 Hematology Analysis 75 3.5.4.2 Analysis of Ferritin and sTfR 76 3.5.5 Assignment of Iron Status 78 3.5.6 Statistical Analysis 79 3.5.7 Dissemination of Results 83 CHAPTER 4 RESULTS 4.1 Study Population 85 4.1.1 Participant Recruitment 85 4.1.2 Nutrition Research Clinics and Attendance 87 4.1.3 Description of Study Participants 89 4.1.3.1 Characteristics of Study Participants 88 4.1.3.2 Feeding Practices of Study Participants 93 4.2 Iron Status of Study Participants 96 4.2.1 Prevalence of Indices of Iron Status Indicative of Iron Deficiency Anemia and Low Iron Stores Among Infants 8-26 Mths of Age 96 4.2.2 Prevalence of Iron Deficiency Anemia and Low Iron Stores among Infants Classified by Ancestry 100 4.3 Relation of Feeding History Determined by the Socio-Cultural and Infant Feeding Questionnaire of Infants 8-26 Mths of Age to Iron Status and Ancestry 104 4.3.1 Infant Feeding History as Reported on the Socio-Cultural and Infant Feeding Questionnaire Among Infants Grouped by Iron Status 103 4.3.2 Frequency of Infant Feeding History Associated with Risk of Iron Deficiency Anemia and Low Iron Stores Among Infants from Caucasian and Chinese Ancestries 108 4.4 Relation of the Intakes of Major Food Sources of Iron and Dietary Factors Influencing Iron Absorption Determined by the FFQ Among Infants 8-26 Mths of Age to Iron Status and Ancestry Ill iv Table of Contents 4.4.1 Intakes of Major Food Sources of Iron and Dietary Factors Influencing Iron Absorption Determined by FFQ Among Infants Grouped by Iron Status Ill 4.4.2 Intake of Foods Providing Major Food Sources of Iron and Factors Influencing Iron Absorption Determined by the FFQ Among Infants from Caucasian and Chinese Ancestries 118 4.5 Relation of Estimated Intake of Iron and Factors Known to Influence Iron Absorption Determined from Non-Milk Foods Determined by 3d-FR of Infants 8-26 Mths of Age to Iron Status and Ancestry 124 4.5.1 Intakes of Iron and Dietary Factors Known to Influence Iron Absorption From Non-Milk Foods Determined by 3d-FR Among Infants Grouped by Iron Status 129 4.5.2 Intakes of Iron and Dietary Factors Known to Influence Iron Absorption Determined by 3d-FR Among Infants from Caucasian and Chinese Ancestries 129 4.6 The Value of the 3-day Food Record and Food Frequency Questionnaire in Predicting Biochemical Indices of Iron Status Among Infants 8-26 Mths of Age 134 4.7 Development of a Screening Tool to Predict Infants at Risk for Iron Deficiency Anemia and Low Iron Stores 144 4.7.1 Multivariate Predictors of Iron Deficiency Anemia and Low Iron Stores Among Study Participants 144 4.7.2 Classification and Regression Tree (CART) Analyses of Predictors of Risk for Iron Deficiency Anemia and Low Iron Stores Among Study Participants. 147 4.8 Comparison of Dietary Parameters as Determined by the 3-day Food Record and Food Frequency Questionnaire 152 4.9 Clinical Utility of the Transferrin Receptor for Detecting Iron Deficiency Anemia and Low Iron Stores Among Infants 8-26 Mths of Age 155 4.10 Summary of Findings 163 4.10.1 Study Participants 163 4.10.2 Summary of Results with Regard to Hypotheses 161 4.10.3 Relative Validity of FFQ Compared with 3d-FR. 167 4.10.4 Multivariate Predictors of Poor Iron Status 168 4.10.5 Clinical Utility of the sTfR. 168 CHAPTERS DISCUSSION 5.1 Iron Status of Caucasian and Chinese Infants Aged 8-26 Mths in Vancouver 169 5.1.1 Prevalence of Iron Deficiency Anemia and Low Iron Stores Among Caucasian and Chinese Infants Aged 8-26 Mths in Vancouver 170 5.1.2 Implications of the High Prevalence of Iron Deficiency Anemia and Low Iron Stores at 8-12 Mths of Age and Low Iron Stores at 13-26 Mths of Age for Infant Health and Development 175 5.2 Strategies for Identification of Infants at Risk for Iron Deficiency Anemia 180 5.2.1 Use of Dietary Assessment Instruments to Assess Iron Status in Infancy 180 5.2.1.1 Value of the Food Frequency Questionnaire for Assessing the Intake of Iron and Other Factors Influencing Iron Absorption. 180 v Table of Contents 5.2.1.2 Value of Assessment of Feeding History and Current Dietary Intake for Classifying Infants by Iron Status 185 5.2.2 Use of sTfR to Assess Iron Status in Infancy 196 5.2.3 Use of a Brief Dietary Assessment Tool versus Biochemical/Hematological Indices of Iron Status as a First Stage Screening Test to Identify Infants at Risk for Iron Deficiency Anemia 201 5.3 Strategies for Primary Prevention of Iron Deficiency Anemia in Infancy: Are Current Guidelines for Infant Feeding Effective in Preventing Iron Deficiency Anemia in Chinese and Caucasian Infants? 204 5.4 Study Limitations 207 5.5 Conclusions and Implications for Measurement, Policy and Practice 211 5.6 Future Directions 217 REFERENCES 221 APPENDICES Appendix A. Certificate of Ethical Approval, University of British Columbia 241 Appendix B. Socio-Cultural and Infant Feeding Questionnaire 242 Appendix C. 3-day Food Record (3d-FR) Package 255 Appendix D. Food Frequency Questionnaire (FFQ) for Parents of Infants 8-26 Mths of Age. 264 Appendix E. Example of a Food List for FFQ Average 289 Appendix F. List of USER Codes for all Foods for which Food Composition Data was added to the ESHA Database 292 Appendix G. Socio-Cultural and Infant Feeding Questionnaire (Chinese) 303 Appendix H. 3-day Food Record (3d-FR) Package (Chinese) 316 Appendix I. Food Frequency Questionnaire (FFQ) for Parents of Infants 8-26 Mths of Age 321 (Chinese) Appendix J. Recruitment Letter (English) 348 Appendix K. Recruitment Letter (Chinese) 349 Appendix L . Personal Data Form 351 Appendix M . Consent Forms 352 Appendix N. Coded Food Frequency Questionnaire (FFQ) for Parents of Infants 8-26 Mths of Age 358 Appendix O. FFQ Food Categories used to Catagorize Foods for Data Analysis 383 vi Table of Contents Appendix P. Feedback Letter and Study Information Pamphlets 384 Appendix Q. Results 397 vii List of Tables List of Tables Table 2.1. Distribution of iron in the body 15 Table 2.2. Changes in body iron during infancy during the first year of life in a hypothetical infant and estimated requirements for endogenous and dietary iron 33 Table 2.3. Estimated amount of iron from solid foods needed to meet the endogenous iron requirement of infants 6-24 mths of age in relation to type of primary milk feeding 39 Table 2.4. Summary of studies of iron intake in infancy (U.S.) 41 Table 2.5. Summary of studies of iron intake in infancy (Europe/Asia) 43 Table 2.6. Summary of studies of iron intake in infancy (Canada) 44 Table 2.7. Summary of data reported on the prevalence of iron deficiency anemia and low iron stores among infants in developed countries 45 Table 2.8. Summary of studies on iron deficiency anemia and low iron stores among full-term infants from Caucasian ancestries 50 Table 2.9. Summary of studies of iron deficiency anemia and low iron stores among full-term infants from Chinese ancestries 52 Table 3.1. Summary of cut-off values used to classify iron status of infants participating in the study... 79 Table 4.1 Summary of participant recruitment from birth lists 86 Table 4.2. Clinic locations and number of infants who attended 88 Table 4.3. Demographic and socio-cultural characteristics of study participants 90 Table 4.4. Self-reported family food practices 92 Table 4.5. Feeding practices of study participants grouped as 8-12 and 13-26 months of age as reported on the Socio-Cultural and Infant Feeding Questionnaire 94 Table 4.6. History of use of vitamin and/or mineral supplements among study participants as reported on the FFQ 95 Table 4.7. Hematological and biochemical indices of iron status among infants participating in the study and the percentage of infants with biochemical indices below cut-off points 97 Table 4.8. Number of infants with iron deficiency anemia, low iron stores, normal iron status and low hemoglobin 99 Table 4.9. Hematological and biochemical indices of iron status among Caucasian and Chinese infants and the percentage of infants with biochemical indices below normal cut-off points 101 Table 4.10. Number of infants with iron deficiency anemia, low iron stores, normal iron status, and low hemoglobin grouped by ancestry and age 103 Table 4.11. Summary of feeding history reported on the Socio-Cultural and Infant Feeding Questionnaire among infants 8-12 mths of age grouped by iron status 106 Table 4.12. Summary of feeding history reported on the Socio-Cultural and Infant Feeding Questionnaire among infants 13-26 mths of age grouped by iron status 107 Table 4.13. Frequency of feeding history associated with risk for iron deficiency among infants 8-12 mths of age from Caucasian and Chinese ancestries 109 Table 4.14. Frequency of feeding history associated with risk for iron deficiency among infants 13-26 mths of age from Caucasian and Chinese ancestries 110 Table 4.15. Median daily intakes of food and iron from different food categories determined by the FFQ for infants 8-12 mths of age classified according to iron status 113 Table 4.16. Median daily intakes of food and iron from different food categories determined by the FFQ for infants 13-26 mths of age classified according to iron status 114 Table 4.17. Median daily intakes of food and iron from different food categories determined by the FFQ for infants 8-12 mths of age from Caucasian and Chinese ancestries 120 Table 4.18. Median daily intakes of food and iron from different food categories determined by the FFQ for infants 13-26 mths of age from Caucasian and Chinese ancestries 121 Table 4.19. Intakes of iron from non-milk foods determined by the 3d-FR among infants with poor iron status and normal iron status who were breast-fed or fed low iron "milk", or iron supplemented 126 Table 4.20. History of use of supplements among study participants grouped by iron status 127 Table 4.21. Intakes of energy, heme iron, non-heme iron, vitamin C, calcium and dietary fibre from non-milk foods determined by the 3d-FR among infants 8-12 mths of age grouped by iron status 128 Table 4.22. Intakes of energy, heme iron, non-heme iron, vitamin C, calcium and dietary fibre from non-V l l l List of Tables milk foods determined by the 3d-FR among infants 13-26 mths of age grouped by iron status 128 Table 4.23. Intakes of iron from non-milk foods determined by 3d-FR among infants from Caucasian and Chinese ancestries grouped by age and whether or not they had been breast-fed/low iron "milk" fed or had received supplemental iron 131 Table 4.24. History of supplement use among Caucasian and Chinese infants 132 Table 4.25. Intakes of energy, non-heme iron, heme iron, vitamin C, calcium and dietary fibre from non-milk foods determined by 3d-FR among infants 8-12 mths of age from Caucasian and Chinese ancestries. 133 Table 4.26. Intakes of energy, non-heme iron, heme iron, vitamin C, calcium, and dietary fibre from non-milk foods determined by 3d-FR among infants 13-26 mths of age from Caucasian and Chinese ancestries 133 Table 4.27. Classification of infants by quartiles of total iron intake as determined from a 3d-FR and FFQ compared with classification by quartiles of sTfR, serum ferritin and sTfR: ferritin 143 Table 4.28. Logistic regression analysis of low iron stores without or with iron deficiency anemia, with infant age and with feeding practices 145 Table 4.29. Logistic regression analysis of low iron stores with infant age and with feeding practices 146 Table 4.30. Sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values of specific cut-off levels of predictive values of dietary predictors of risk compared with the diagnosis of iron deficiency anemia or low iron stores 148 Table 4.31. Comparison of the intakes of energy, total iron, heme iron, non-heme iron, vitamin C, calcium, and dietary fibre from non-milk foods determined by the FFQ and 3d-FR analyses in infants 8-26 mths of age 153 Table 4.32. Comparison of intakes of food from the food groups that provide major sources of iron and dietary factors influencing iron absorption determined by the FFQ and 3d-FR analyses in infants 8-26 mths of age 154 Table 4.33. Soluble transferrin receptor (sTfR) and sTfR:ferritin ratio among infants aged 8-26 mths with normal iron status, low iron stores and iron deficiency anemia 157 Table 4.34. Soluble transferrin receptor (sTfR) and sTfR:ferritin ratio in infants aged 8-26 mths with normal iron status 157 Table 4.35. Soluble transferrin receptor (sTfR) and sTfR:ferritin ratio in infants with iron deficiency anemia 159 Table 4.36. Sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values of specific cut-off values of the soluble transferrin receptor (sTfR) compared with diagnosis of iron deficiency anemia defined on the basis of Hgb and serum ferritin, and low iron stores defined on the basis of serum ferritin 162 Table A . l . Hematological and biochemical indices of iron status among infants participating in the study 397 Table A.2. Infants with iron deficiency anemia, normal iron status, and low hemoglobin when grouped by gender 398 Table A.3 . Laboratory indices used to define iron status among study participants grouped as normal iron status, low iron stores, iron deficiency anemia and low hemoglobin 408 Table A.4. Maternal socio-cultural background of study participants 410 ix List of Figures LIST OF FIGURES Figure 2.1. Characteristic stages in the development of iron deficiency anemia, schematic changes in selected indices of iron status and typical laboratory profile in infants 19 Figure 2.2. Schematic representation of the determinants of iron balance during infancy 26 Figure 2.3. Schematic illustration of the major sources of dietary iron, and inhibitors and enhancers of iron absorption, shown with reference to the recommended patterns of food consumption during infancy. 35 Figure 4.1. Estimated contribution of food groups to the total intake of food (g/100 g/day) determined by FFQ among infants 8-12 mths of age who had poor iron status or normal iron status 116 Figure 4.2. Estimated contribution of iron by food groups to the total iron intake (mg/100 mg/day) determined by FFQ among infants 8-12 mths of age who had poor iron status or normal iron status 116 Figure 4.3. Estimated contribution of food groups to the total intake of food (g/100 g/day) determined by FFQ among infants 13-26 mths of age who had poor iron status or normal iron status 117 Figure 4.4. Estimated contribution of iron by food groups to the total iron intake (mg/100 mg/day) determined by FFQ of infants 13-26 mths of age who had poor iron status or normal iron status 117 Figure 4.5. Estimated contribution of food groups to the total intake of food (g/100 g/day) determined by FFQ among infants 8-12 mths of age from Caucasian and Chinese ancestries 122 Figure 4.6. Estimated contribution of iron by food groups to the total iron intake (mg/100 mg/day) as determined by FFQ among infants 8-12 mths of age from Caucasian and Chinese ancestries 122 Figure 4.7. Estimated contribution of food groups to the total intake of food (g/100 g/day) as determined by FFQ among infants 13-26 mths of age from Caucasian and Chinese ancestries 123 Figure 4.8. Estimated contribution of iron by food groups to the total iron intake (mg/100 mg/day) as assessed by FFQ among infants 13-26 mths of age from Caucasian and Chinese ancestries 123 Figure 4.9. Distribution of infants with poor iron status and normal iron status with iron intakes determined by 3d-FR expressed as a percent of the RNI 127 Figure 4.10. Distribution of Caucasian and Chinese infants with iron intakes determined by 3d-FR expressed as a percent of the RNI 132 Figure 4.11. Scatterplots of hemoglobin (g/L) versus intakes of total iron (mg/day) and heme iron (mg/day) as estimated from a. 3-day food record (3d-FR), n=139 and b. Food Frequency Questionnaire (FFQ), n=140 135 Figure 4.12. Scatterplots of serum ferritin (ug/L) versus intakes of total iron (mg/day) and heme iron (mg/day) as estimated from a. 3-day food record (3d-FR), n=138 and b. Food Frequency Questionnaire (FFQ), n=140 137 Figure 4.13. Scatterplots of sTfR (nmol/L) versus intakes of total iron (mg/day) and heme iron (mg/day) as estimated from a. 3-day food record (3d-FR), n=138 and b. Food Frequency Questionnaire (FFQ), n=140 139 Figure 4.14. Scatterplots of sTfR:ferritin versus intakes of total iron (mg/day) and heme iron (mg/day) as estimated from a. 3-day food record (3d-FR), n=138 and b. Food Frequency Questionnaire (FFQ), n=140 141 Figure 4.15. Receiver Operating Characteristic (ROC) Curve for dietary predictors to detect infants with iron deficiency anemia or low iron stores 149 Figure 4.16. Classification and Regression Tree (CART) analysis of dietary predictors to detect infants with poor iron status 150 Figure 4.17. Simplified Classification and Regression Tree (CART) analysis of dietary predictors to detect infants with poor iron status 151 Figure 4.18. Histogram and fitted Gaussian distribution curves of a. sTfR and b. sTfR:ferritin ratios in infants aged 8-26 mths with normal iron status, n=94 158 Figure 4.19. Scatterplots of relations between sTfR concentration and a. ferritin and b. hemoglobin in infants 8-26 mths of age, n= 140 160 Figure 4.20. Scatterplot of the relation between sTfR:ferritin ratio and hemoglobin in infants 8-26 mths of age, n= 140 161 Figure A . l . Scatterplot of energy intake from non-milk foods (kcal/day) as estimated from a 3d-FR versus age among infants 8-26 mths of age, n = 146 399 Figure A.2. Scatter plot of total iron intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants grouped by iron status, n = 146 399 x List of Figures Figure A.3. Scatterplot of heme iron intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants aged 8-26 mths, n = 146 400 Figure A.4. Scatter plot of non-heme iron intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants aged 8-26 mths, n = 146 400 Figure A.5. Scatterplot of vitamin C intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants aged 8-26 mths (n =146, r = 0.58, p<0.001) 401 Figure A.6. Scatterplot of calcium intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants aged 8-26 mths, n = 146 401 Figure A.7. Scatterplot of dietary fibre intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants aged 8-26 mths, n = 146 402 Figure A.8. Scatterplots of energy intake from non-milk foods (kcal/day) as estimated from a 3d-FR versus age among infants of Caucasian (n =78) and Chinese (n =47) ancestry 402 Figure A.9. Scatterplots of total iron intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants of Caucasian (n = 78) and Chinese (n = 47) ancestrytnon-iron supplemented, • iron supplemented 403 Figure A.10. Scatterplots of heme iron intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants of Caucasian (n = 78) and Chinese (n = 47) ancestry 403 Figure A. 11. Scatterplots of non-heme iron intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants of Caucasian (n = 78) and Chinese (n = 47) ancestry, *>non-iron supplemented, • iron supplemented 404 Figure A.12. Scatterplots of vitamin C intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants of Caucasian (n = 78) and Chinese (n = 47) ancestry 404 Figure A.13. Scatterplots of calcium intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants of Caucasian (n = 78) and Chinese (n = 47) ancestry 405 Figure A.14. Scatterplots of fibre intake from non-milk foods (mg/day) as estimated from a 3d-FR versus age among infants of Caucasian (n = 78) and Chinese (n = 47) ancestry 406 Figure A.15. Scatterplots of infants aged 8-26 mtiis grouped by transferrin receptor (sTfR) concentrations and ratio of sTfR to ferritin (sTfR:ferritin) according to assignment of iron status in 148 healthy infants 407 Figure A. 16. Receiver Operating Characteristic Curves (ROC) for sTfR as an indicator of iron deficiency anemia and low iron stores 409 xi Glossary of Terms Glossary of Terms Abbreviations 24-hr recall 24-hour dietary recall 3d-FR 3-day food record 3d-WFR 3-day weighed food record ATP adenosine triphosphate BCCH British Columbia's Children's Hospital CC Community Centre Fe 2 + ferrous Fe 3 + ferric Fe 4 + ferryl FFQ food frequency questionnaire for parents of infants 8-26 months of age GI gastrointestinal Hgb hemoglobin HU Health Unit IDA iron deficiency anemia IDE iron deficiency erythropoiesis M C H mean cell hemoglobin MPF Meats, poultry and fish mths months n number NH Neighbourhood House RBC red blood cell RDA U.S. Recommended Dietary Allowance RNI Canadian Recommended Nutrient Intake SES Socio-economic status sTfR soluble transferrin receptor xii Glossary of Terms TfR transferrin receptor TIBC total iron binding capacity TS transferrin saturation U.S. United States WIC Program Special Supplemental Food Program for Women, Infants and Children Program Definition and Terms Units micrograms g grams kcal kilocalorie L litre mg milligrams mL millilitres Dietary Items food consumption patterns - retrospective information collected by the FFQ on the foods consumed in the 2 weeks preceding the study. infant feeding history - retrospective information on infant feeding practices determined by the Socio-Cultural and Infant Feeding Questionnaire. Questions on infant feeding practices included the following: 1) duration of breast-feeding, 2) the age of introduction and types of infant formula (low iron or iron-fortified), cows' milk (whole, 2%, 1%, or skim) or other milks (goats' milk, soy milk, etc.) fed from birth to the time of the study, 3) the type and amount of infant formula, cows' milk or other milks currently fed, 4) the age of introduction, duration of feeding and, where applicable, reasons for stopping feeding of solid foods including cereals, rice, pasta, breads/crackers, vegetables, fruits, legumes, dairy and other animal products (e.g. eggs, meat, poultry or fish) and fruit juices, 5) the type of infant cereal(s) introduced, the liquid(s) used to prepare infant cereals, and for those who had not yet introduced infant cereal, the reasons for not introducing an infant cereal. Classification of Iron Status iron deficiency/poor iron status - includes both iron deficiency anemia and low iron stores, iron deficiency anemia - Hgb <110 g/L with a ferritin of £12 ug/L. low iron stores - Hgb ^110 g/L but with a serum ferritin of <12 ug/L. xiii Glossary of Terms normal iron status - nonanemic, iron sufficient, Hgb >110 g/L, a serum ferritin >12 u,g/L and a white blood cell count (WBCC) 1^8 xl09/L. TfR - transferrin receptor values based on assays that use cellular disulfide-linked dimer cellular TfR from human placental tissue. Used in the present study. sTfR - soluble transferrin receptor values based on an assay that uses plasma or serum sTfR monomers. Used in the present study. Other Infants - for the purpose of this study includes infants and young children <3£ months. xiv Acknowledgements ACKNOWLEDGEMENTS This research was made possible through funding by the British Columbia Medical Services Foundation and through studentship and research funding by the British Columbia's Health Research Foundatioa Grateful acknowledgement is extended to the Vancouver Health Board, Public Health Nutritionists, Vicki Boere, Barbara Crocker, Corinne Eisler and Helen Yeung, and Nurses for their help throughout the study and their commitment to collaborative research. I owe a deep gratitude to the parents and infants who participated in this study - without them this work would not have been possible. I feel privileged to have been able to cross boundaries to weave my work within the realm of the individual interdisdplinary graduate studies program. I owe deep gratitude to my supervisor, Dr. Sheila Innis, for her contagious passion about her work, and for challenging and supporting me throughout my training. I was also extremely fortunate to have a wonderful committee: to Dr. Bob Armstrong, Dr. Susan Barr, Dr. Jim Frankish and Dr. Sam Sheps and to them I extend my deep gratitude. Along with Dr. Innis, they have supplied a collective and exceptional depth of knowledge, experience, guidance and support along my doctoral path. My interdisdplinary training has been stretched and strengthened by the passionate commitment and ability of Dr. Rhodri Winsor Liscombe, and prior to him, Dr. Laurie Ricou to guide and support students, and foster excellence in interdisciplinary training. I am particularly indebted to my friends, Ryna, Cathy, Loraina, Vikki, Sylvia, Carolanne, Angela, Sandra, Tim, GaiL Dorcas who over the years, have provided support, encouragement, and most importantly lots of laughter and wonderful memories. I was very fortunate and grateful to have worked with an exceptional and dedicated group of students, Loraina, Vikki, Carolanne, Angela, Sandra, Dorcas and Alice and research fellows, Sylvia and Tim, who gave me encouragement, intellectual contributions, and invaluable friendships and support I particularly want to acknowledge the help and trouble shooting from Roger Dyer over the years. I would also like to extend thanks to Sylvia de la Presa, Loraina Stephen, Carolanne Nelson, Paula Waslen, Elbe MacKay, Annie Wong, Vikki Lalarie, Catherine Atchison and Laurie Nicol for their assistance with my research clinics, and to Loraina, Paula and Vivienne for their help on the development of the questionnaires for this study. I am also indebted to the work of numerous summer students and volunteers, most notably, Vivienne Lau, Maria Law, Janice Chan, and Jane Wark, who without their assistance with my questionnaire development and translation, research clinics and endless hours of entry of dietary data, this work would have not have been possible. Many other people deserve thanks for their contributions to this study: the groups at Sheway Community Project Women and xv Acknowledgements Children, UBC Family Housing, and the North and South Health Units for their help with the pilot study, Thomas Lam, for his help with development of the FFQ Analysis database, and Murray MacKinnon for his statistical advice. Finally, I would like to thank the people in my life who mean the most to me, my Mom, Leigh and Gab, my sister Terri and her family, Sterling, Shelby and Carter, and my brother Andy and his fiance, Patricia. Their love, support, encouragement, sacrifices, patience and good humour, and most important, "reality checks" sustained me through my work on this thesis. xvi Dedication DEDICATION Mom and Dad...for you both, with love. Dad, your words provide comfort and strength to me... I know that you are there, always watching over me and believing in me. To Tress, There have been many happiness in my life And that you have been one For I have seen a fine mind come From within a little shell that grows It is my hope that you will find My words of wisdom - for your lines So sit and punch these keys with delight As your essay you have to write For Father's help was gladly given As he types this in the kitchen To see your mark for the work you've been given Will be his reward when he goes to heaven And when to college you must go To seek a life with friends and foe Remember dear old Dad at home The one with whom you argued so Still his thoughts and hopes you'll find Within your heart that may entwine First things first we must agree An essay done for me to see ? ? ? -Dad Your love and the memory of your example, and your aspirations are with me always. Mom, your amazing strength, determination, and ability to sacrifice and give to others have been a source of inspiration and strength throughout my life. You are always there for me... with your love and support, and for that I am so grateful. -Patty xvii Chapter 1. Introduction CHAPTER 1. INTRODUCTION 1.1 Background The plausibility and congruence of the available animal and human evidence strongly suggest that iron deficiency anemia (IDA) has the potential to impair infant health and development IDA in infancy has been associated with impaired growth (Auckett et al., 1986; Briend et al., 1990; Chwang et al., 1988; Latham et al., 1990), immunity (Galan et al., 1992; Thibault et al., 1993), mental (Lozoff et al., 1982, 1987, 1996; Walter et al., 1983, 1989; Idjradmata & Poffitt, 1993; Grindulis et al., 1986) and motor (Lozoff et al., 1982, 1987; Walter et al., 1983; Idjradinata & Poffitt, 1993; Gnndulis et al., 1986) development, behavior (Walter et al., 1983, 1989; Lozoff et al., 19%; Lozoff et al., 1998) and educational performance later in life (Paid et al., 1985; Watkins & PoUitt, 1990; Lozoff et al., 1991; Hurtado et al., 1999). The period beginning at about 4 mths of age to the 2nd year of life is critical for iron balance because iron reserves become depleted at a time when the iron demand for growth and development is high, and there is potential for an inadequate dietary iron supply. Research into potential strategies for prevention of IDA during infancy is, therefore, of considerable public health importance. There has been significant advancement during the last 30 years in our understanding of iron nutrition and in prevention strategies aimed at reducing the prevalence of IDA in infancy (Dallman, 1990; Yip, 1997). Despite this, IDA remains a problem in both developing and developed countries, affecting 25% of infants worldwide (deMaeyer et al., 1985; Scrimshaw, 1991; United Nations, 1989), and up to 10-12% of infants in developed countries (deMaeyer et al., 1985; Stevens, 1991). The national prevalence of IDA among infants in Canada is unknown However, studies have shown that infants from disadvantaged families (Lehmann et al., 1992), Chinese ancestries (Chan Yip & Gray-Donald, 1987) and aboriginal communities (Moffatt et al., 1991; Cruz et al., 1990; Whalen et al., 1997; Sawchuket et al., 1996; Willows et al., 2000), and those breast-fed beyond 3-6 mths of age (Innis et al., 1997; Siimes et al., 1984; Calvo et al., 1992; Pizarro et al., 1991; Walter et al., 1993; Willows et al., 2000) may be particularly vulnerable to developing IDA. Concern has recently been raised that IDA is a significant public health nutrition problem at 9 mths of age, and possibly into the 2nd year of life among infants in Vancouver (Lwanga, 1996; Innis et al., 1997). This is based on a 1993 study in Vancouver that found that the prevalence of IDA and low iron stores was high (8% and 25%, respectively) among Caucasian infants at 9 mths of age, and even higher (15% and 30% respectively) among those breast-fed >8 mths 1 Chapter 1. Introduction (Innis et al., 1997). In contrast to a high prevalence of 11% IDA reported for 6-12 mth old Chinese infants in Montreal (Chan-Yip & Gray-Donald, 1987), the prevalence of IDA was found to be low, affecting 4% of 9 mth old Chinese infants in Vancouver (Lwanga, 1996; Innis et al., 1997). The work by Innis et al. (1997) found that higher rates of breast-feeding were associated with a higher risk of IDA among Caucasian infants, while higher rates of feeding iron-fortified infant formula were associated with a lower risk of IDA among Chinese infants. The only other published data on the iron status of Chinese infants in Canada, that by Chan-Yip & Gray-Donald (1987), suggested that the prevalence of IDA may increase from the first to the 2nd year of life among Chinese infants. It has been documented that while the majority of Caucasian infants are fed iron-fortified infant cereal by 4-6 mths of age (Greene-Finestone et al., 1991; Zlotkin et al., 1981; Ernst et al., 1990), Chinese infants are often fed congee as their first complementary food in place of iron-fortified infant cereals, and given cows' milk early and in large quantities (Leung & Davis, 1994; Li, 1985; Chan-Yip & Gray-Donald, 1987; Hui, 1997). It is possible that the introduction of a variety of complementary foods, including iron-fortified infant cereal may decrease the risk of IDA among Caucasian infants in the 2n d year of life. Weaning practices, such as the use of congee, which tends to have low iron bioavailability, and excessive intakes of cows' milk, on the other hand, may increase the risk of IDA in the 2n d year among Chinese infants. Previous studies have reported that the majority of infants in Vancouver are introduced to iron-containing solid foods at appropriate ages (Williams et al., 1996). However, studies concerning infant feeding practices among infants in Canada have not obtained quantitative data on dietary intakes. Thus, information is lacking on the risk of IDA and low iron stores among Caucasian and Chinese infants from the latter half of the first year throughout the 2nd year of life with respect to feeding practices, dietary intakes and sources of iron and other factors influencing iron absorption, and the overall composition of the weaning diet Since 1979, the primary prevention of IDA in Canada has focused on the fortification of infant cereals and formulas with iron, and on the education of parents and medical professionals to promote feeding practices thought to prevent IDA. Specifically, these feeding practices are to exclusively breast-feed for at least 4 mths, use iron-fortified formula as a breast milk substitute, introduce iron-fortified infant cereals at 4-6 mths of age, delay introduction of cow's milk until 9-12 mths of age, and continue use of iron-fortified foods beyond one year of age (Canadian Pediatric Society (CPS) Nutrition Committee, 1979 & 1991; CPS, Dietitians of Canada and Health Canada, 1998). Actual infant feeding practices in Canada are now more reflective of infant feeding recommendations than the practices reported 30 years ago (Health and Welfare Canada, 1993, Health and Welfare 2 Chapter 1. Introduction Canada, 1991; MacNally et al., 1985; Tanaka et al., 1987; Williams et al., 1996; Kwavnick et al., 1999). Despite the availability of iron-fortified infant foods and an overall improvement in feeding practices among Canadian infants, high rates of IDA continue to occur among vulnerable subgroups of infants. Clearly, the strategies currently being used in Canada to prevent IDA are not effective among subgroups of infants. There is a lack of information on which to base dietary recommendations for the prevention of IDA in infancy (Oski, 1993). For example, the Canadian Recommended Nutrient Intake (RNI) for iron is based on the theoretical estimated need for iron and does not take into consideration infant diets that may be low in heme iron or of low iron bioavailability. Although the primary strategy for the prevention of IDA among breast-fed infants is the introduction of iron-fortified infant cereals at 4-6 mths of age, cross-sectional studies of actual cereal intakes among infants suggest that this recommendation may not be efficacious (Zlotkin et al., 1981; Gerber Infant Nutrition Survey, 1989). Studies are needed to develop and investigate alternative strategies aimed at the prevention of IDA among infants from disadvantaged families, Chinese ancestries, aboriginal communities, and those breast-fed beyond 3-6 mths of age. Current data on dietary intakes, sources of iron and other factors influencing iron absorption for infants in Canada throughout the weaning period, however, is lacking. Traditionally, studies that have assessed iron nutrition in infancy have relied on dietary records and 24-hr recalls. Research in infant nutrition has been hampered by a lack of published studies with parents of infants which have validated dietary assessment tools, such as food frequency questionnaires (FFQs) that pose less respondent burden and cost than diet records and 24-hr recalls. FFQs for assessing iron intake have been developed and validated for use with adults (Willett, 1989), but studies on the validation of a FFQ for assessing iron nutrition during infancy and throughout the weaning period have not been published. Further advances in the prevention of IDA in infants might be achieved with instruments, such as an FFQ, that provides a rapid and easily administered means of assessing the dietary intakes and sources of iron and inhibitors and enhancers of iron absorption. Further advances in the prevention of IDA in infancy might also be achieved by improvements in strategies for early detection. Infants in Canada with IDA are currently identified using a case-finding approach, whereby routine blood work including a complete blood count (CBC) is done, either if IDA is suspected or if the infant is being investigated for reasons unrelated to IDA. In cases where abnormal red blood cells (RBC) indicative of anemia are found, the diagnosis of iron deficiency may be confirmed with investigation of serum ferritin, or by noting a positive response to iron therapy (Canadian Task Force for Periodic Health Examination, 1994). The invasiveness and unacceptability of a blood test for parents of the otherwise healthy infant have been documented 3 Chapter 1. Introduction (Mills, 1990; James et al., 1997). The Canadian Task Force for Periodic Health Examination (1994) concluded that there is insufficient evidence to recommend screening normal infants for IDA with a blood test. The idea of a first stage dietary screening tool to assess the diets of infants likely to be at risk for IDA to determine the need for a 2n d stage screening with a blood test is a possible alternate strategy. Although the Canadian Task Force for Periodic Health Examination has recommended this since 1994, only 2 studies have evaluated the use of a dietary screening tool for predicting the risk of IDA in infancy (Boutry & Needlman, 1996; Bogen et al., 2000). No standardized dietary instruments for assessing the diets of Caucasian or Chinese infants for iron nutrition, or predicting risk of poor iron status are as yet available. Further, specific dietary factors that can be used in a practical dietary based screening tool to predict poor iron status in Caucasian and Chinese infants have not been published. Advances in the laboratory assessment of iron status in infancy might also achieve further improvements in strategies for early detection of IDA. Previous studies with adults (Skikne et al., 1990; Heubers et al., 1990; Kohgo et al., 1987; Ferguson et al., 1992), pregnant women (Carriaga et al., 1991) and children (Punnonen et al., 1994) suggest the potential value of measures of the soluble transferrin receptor (sTfR) for identifying infants with early iron deficiency erythropoiesis (TDE). sTfR may be particularly valuable for both diagnostic and screening purposes because, unlike ferritin and other laboratory indices of iron status, sTfR is not falsely elevated by infection (Ferguson et al., 1992; Punnonen et al., 1994; Pettersson et al., 1994; Thorstensen & Ramsio, 1993), and requires a small sample size (10 uL). Information on the use of sTfR for assessing iron status in infancy, however, is limited (Virtanen et al., 1999; Yeung & Zlotkin, 1997; Lonnerdal & HemelL 1994; Kuiper-Kramer et al., 1998). Yeung & Zlotkin (1997) have provided valuable data on sTfR concentrations in infants 9 to 15 mths of age, and established reference standards based on an assay system calibrated against cellular TfR. Information on sTfR in infants older than 15 mths of age, and using a more recently available assay system based on natural plasma sTfR, however, have not been published. 4 Chapter 1. Introduction 1.2 Purpose of Study The overall purpose of this cross-sectional study was to determine whether dietary assessment instruments could be used to categorize infants as having normal or poor (i.e. low iron stores or iron deficiency anemia) iron status. Dietary assessment instruments were developed and used to assess the feeding histories and the dietary intakes and sources of energy, iron, and other factors influencing iron absorption of infants aged 8 to 26 mths from Chinese and Caucasian ancestries in Vancouver. The iron status of this group of infants was determined by hematological and biochemical indices of iron status, and related to their retrospective assessment of feeding history, concurrent measures of dietary intakes, and socio-cultural background. This information was then used to make recommendations for strategies for secondary and primary prevention of iron deficiency anemia 5 Chapter 1. Introduction 1.3 Study Objectives The objectives of this study were: 1. To use a 3-day Food Record (3d-FR), food frequency questionnaire (FFQ) and FFQ Analysis Database to determine the dietary sources and intakes of energy, iron (total, heme and non-heme) and major dietary factors likely to affect iron absorption in infants aged 8-26 mths from Caucasian and Chinese ancestries in Vancouver, B.C. 2. To use a Socio-Cultural and Infant Feeding Questionnaire to determine infant feeding histories in infants 8-26 mths of age from Caucasian and Chinese ancestries in Vancouver, B.C. 3. To determine iron status among infants aged 8-26 mths from Chinese and Caucasian ancestries for whom dietary data were collected by concurrent measures of hematological (hemoglobin) and biochemical (serum ferritin, soluble transferrin receptor (sTfR) and sTfR:ferritin) indices of iron status. 4. To explore the relations between iron status of infants classified as having iron deficiency anemia, low iron stores or normal iron status and infant feeding histories, dietary sources and intakes of energy, iron (total, heme and non-heme) and major dietary factors likely to affect iron absorption, and socio-cultural background. 5. To determine which dietary variables as assessed by the Socio-Cultural and Infant Feeding Questionnaire, 3d-FR and FFQ were the best predictors of poor iron status (IDA or low iron stores) among infants aged 8-26 mths from Caucasian and Chinese ancestries. 6. To explore the relation between biochemical indices of iron status (Hgb, serum ferritin, sTfR and sTfR: ferritin) and measures of iron (total and heme) intake in infants aged 8-26 mths from Chinese and Caucasian ancestries. 6 Chapter 1. Introduction 7. To explore the validity of a FFQ for assessing iron nutrition among Chinese and Caucasian infants aged 8-26 mths by comparison with a 3d-FR and biochemical indices of iron status. 8. To determine the distribution of sTfR and sTfRferritin concentrations and the utility of the sTfR as a measure for detecting iron deficiency anemia and low iron stores in infants aged 8-26 mths. 7 Chapter 1. Introduction 1.4 STUDY HYPOTHESES For the purpose of this research the null hypotheses were: 1. There is no difference in the prevalence of iron deficiency anemia and low iron stores between infants 8-12 or 13-26 mths of age in Vancouver of Caucasian compared with Chinese ancestry. 2. Dietary assessment using a Socio-Cultural and Infant Feeding Questionnaire will find no difference in the feeding histories (i.e. the duration of breast-feeding, age of introduction of cows' milk, feeding with iron-fortified infant formula, or use of iron supplements, and age of introduction or duration of feeding of iron-fortified infant cereal or meats) among infants with normal iron status and poor iron status at 8-12 or 13-26 mths of age. 3. Dietary assessment using a food frequency questionnaire (FFQ) will find no difference in the intakes of meat, poultry and fish (MPF), mixed dishes with MPF, iron-fortified infant formula or iron-fortified infant cereal, cows' milk and milk products, soy-based products or regular infant formula among infants with normal iron status and poor iron status at 8-12 or 13-26 mths of age. 4. Dietary assessment using a 3d-FR will find no difference in the intakes of iron (total, heme or non-heme), energy, vitamin C, calcium, or fibre from non-milk foods among infants with poor iron status and infants with normal iron status. 5. Dietary assessment using a Socio-Cultural and Infant Feeding Questionnaire will find no difference in the feeding histories (i.e. the duration of breast-feeding, age of introduction of cows' milk, feeding with iron-fortified infant formula, or use of iron supplements, and age of introduction or duration of feeding of iron-fortified infant cereal or meats) between infants of Caucasian and Chinese ancestry at 8-12 or 13-26 mths of age. 8 Chapter 1. Introduction 6. Dietary assessment using a FFQ will find no difference in the intakes of MPF, mixed dishes with MPF, iron-fortified infant formula, iron-fortified infant cereal, cows' milk and milk products, soy-based products or regular infant formula between infants of Caucasian and Chinese ancestry at 8-12 or 13-26 mths of age. 7. Dietary assessment using a 3d-FR will find no difference in the intakes of iron (total, heme or non-heme), energy, vitamin C, calcium, or fibre from non-milk foods between infants of Caucasian and Chinese ancestry at 8-12 or 13-26 mths of age. 8. Dietary assessment using a FFQ will find no relation between the intakes of total or heme iron and the biochemical indices of iron status, serum ferritin, sTfR and sTfRiferritin, among infants 8-26 mths of age. 9 Chapter 2. Literature Review CHAPTER 2. LITERATURE REVIEW 2.1 Introduction The present study was designed to investigate whether dietary assessment could be used to identify infants with poor iron status. Numerous studies have identified dietary risk factors for poor iron status in infancy (e.g. Pizarro et al., 1991; Innis et al., 1997; Greene-Finestone et al., 1991; Requejo et al., 1999; Mira et al., 1996). The value of dietary assessment instruments to predict the risk of poor iron status in Caucasian and Chinese infants, however, has not been investigated. Dietary assessment instruments able to measure the intake of iron and other dietary factors influencing iron absorption and to predict risk of poor iron status could have considerable value for further advances in research in iron nutrition and prevention of iron deficiency anemia (IDA) in infancy. If the dietary patterns associated with poor iron status among Caucasian and Chinese infants can be identified, then initiatives both to detect infants with these patterns, and to modify the feeding practices associated with risk can be applied in strategies for primary and secondary prevention The following literature review provides a background on the continuing problem of IDA among certain vulnerable subgroups of infants in developed countries, and the implications of poor iron status on infant health and development. Iron balance in infancy is examined in detail in relation to the changes that occur in the functional, transport and storage iron compartments from normal iron status to the development of IDA and to the dietary and non-dietary determinants of iron balance. To better understand the potential use of dietary factors to identify infants at risk for IDA the studies that have examined the amount and bioavailability of iron provided by the primary milk feedings and complementary foods, and the relations between characteristics of the diet in infancy and risk for poor iron status are reviewed in detail. The final section of this literature review examines the effectiveness of current strategies aimed at prevention of IDA among infants in Canada, and identifies the potential value of dietary assessment instruments to improve prevention of IDA and research in the area of iron nutrition in infancy. 10 Chapter 2. Literature Review 2.2 The Importance of Iron in Human Nutrition IDA is the most prevalent single micronutrient deficiency among infants worldwide (deMaeyer et al., 1985; Scrimshaw, 1991; United Nations, 1989). Because IDA has the potential to impair infant health and development, it is of considerable public health importance. The national prevalence of IDA in infants in Canada is unknown. Numerous studies, however, have shown that IDA is a substantial problem among specific infant populations, including infants from families of low socio-economic status (SES) (Lehmann et al., 1992), Chinese ancestries (Chan-Yip & Gray-Donald, 1987) and aboriginal communities (Moffatt et al., 1991; Cruz et al., 1990; Whalen et al., 1997; Sawchuket et al., 1996; Willows et al., 2000), as well as infants breast-fed over 3-6 mths of age not given supplemental iron (Innis et al., 1997; Siimes et al., 1984; Calvo et al., 1992; Pizarro et al., 1991). The risk of IDA is especially high during the latter half of the first year and into the 2n d year of life because the dietary iron intake may be inadequate to meet the high needs for growth and red blood cell (RBC) synthesis (Dallman, 1986). Iron is an essential nutrient that is vital to the maintenance of normal physiological function. Iron is a component of, or cofactor for hundreds of proteins and enzymes (Beard et al., 1996), and as a result, iron deficiency affects many metabolic and enzymatic processes including oxygen transport, oxidative metabolism and cellular growth (Bothwell, 1995; Lynch, 1997). A deficiency of iron is of particular concern in infancy because in addition to the effects of the anemia, iron deficiency in infants is associated with a cascade of nonhematologic consequences, some of which may have a detrimental and irreversible impact on the central nervous system (CNS) (Dallman et al., 1978). An adequate supply of iron is particularly critical during the period of development spanning the 2n d trimester of gestation to 18-24 mths of age. During this period, growth and CNS development (including brain growth, dendritic aborization and myelination) occur at more rapid rates than any other time in life (Dobbing, 1990). A deficiency of iron during this critical period of development places the infant at risk for impaired developmental outcomes (Lozoff et al., 1982, 1987, 1996; Walter et al., 1983, 1989; Grindulis et al., 1986; Moffatt et al., 1994; Williams et al., 1999) and educational performance (Palti et al., 1985; Watkins & Polhtt, 1990; Lozoff et al., 1991; Hurtado et al., 1999). All 7 of the available studies that included careful definitions of iron status and appropriate comparison groups reported that infants with IDA scored lower on tests of mental development before treatment when compared with iron replete age matched controls (Lozoff et al., 1982, 1987, 1996; Walter et al., 1983, 1989; 11 Chapter 2. Literature Review Idjradinata & Pollitt, 1993; Grindulis et al., 1986). Five of the latter studies also reported lower scores on tests of motor development (Lozoff et al., 1982, 1987; Walter et al., 1983; Idjradinata & Pollitt, 1993; Grindulis et al., 1986). Studies that have assessed infant behavior have reported that infants with IDA present with wariness, hesitancy, tiredness, inattentiveness, decreased activity and general lack of involvement with testing stimuli (Walter et al., 1983, 1989; Lozoff et al., 1996 & 1998). It is possible that the association between IDA and lower developmental test scores may be due to other coexisting factors, such as poor social and economic conditions, or other co-existing nutrient deficiencies or toxicities. Nonetheless, it is highly plausible that a deficiency of iron could delay both mental and motor development because iron plays many roles in CNS function These involve the role of iron in dopamine metabolism (Nelson et al., 1997; Ashkenazi et al., 1982), and in synthesis of lipid components of the myelin sheaths (Connor & Menzies, 1996). Further, a brief period of severe IDA in the young, but not the adult rat has been reported to result in deficits in brain iron and learning capacity that were not corrected by iron therapy (Yehuda & Youdim, 1989; Erikson et al., 1997). Felt & Lozoff (1996) demonstrated that these effects were more pronounced in neonatal rats who developed iron deficiency in the suckling period than in the fetal period. Moreover, randomized controlled trials have demonstrated that iron supplementation with iron-fortified infant formula in place of unmodified cows' milk can prevent IDA and its associated declines in psychomotor development in the 2n d half of the first year of life (Moffatt et al., 1994; Williams et al., 1999). Evidence from epidemiological and experimental studies, however, suggests that IDA is frequently only one of many important, often co-existing factors that may result in impaired infant development Despite the possible confounding by socio-environmental factors, such as the family environment, information from all (Lozoff et al., 1989 & 1996; Grindulis et al., 1986; Walter et al., 1983 & 1989) except one study (Idjradinata & Pollitt, 1993) have provided evidence that treatment of IDA in infancy does not fully reverse the delays in cognitive development, i.e. language acquisition and abstract thinking. The study of Idjradinata & Pollitt (1993) found that supplementation of 13-14 mth old Indonesian infants with a hemoglobin (Hgb) of <105 g/L with 3 mg ferrous sulfate/day for 3 mths both reversed the anemia and low developmental test scores. Both Costa Rica and Chile have relatively high standards of living and are more developed than Indonesia. Thus, the infants studied by Lozoff et al. (1996) in Costa Rica and Walter et al. (1989) in Chile were probably from higher SES family backgrounds than the infants studied by Idjradinata & Pollitt (1993) in Indonesia Further, the infants studied by Idjradinata & Pollitt (1993) had lower pretreatment scores relative to the nonanemic infants than the infants in the 12 Chapter 2. Literature Review 2 former studies, and therefore, may have been more vulnerable to the nonhematological consequences of the IDA and had more room for improvement. While the study by Idjradinata & Pollitt (1993) suggests that the identification and treatment of anemia in infancy may be effective for preventing of the potential detrimental long-term consequences of IDA in some infants if the anemia is corrected early and is of relatively short duration, other studies suggest that detection and treatment of EDA may not be effective in all infants or under all circumstances (Lozoff et al., 1989 & 1996; Grindulis et al., 1986; Walter et al., 1983 & 1989). Lozoff et al. (1996) reported that lower mental developmental test scores in infants 12-23 mths of age with IDA persisted despite correction of the anemia. Consistent with the findings of Lozoff et al. (1996), long-term follow up studies of children treated for EDA as infants found lower test scores in mental and motor assessments up to 5-10 years later when compared with children without a history of IDA, even after adjustment for covariates (Paid et al., 1985; Watkins & Pollitt, 1990; Lozoff et al., 1991; Hurtado et al., 1999). However, the possibility cannot be discounted that differences in other environmental factors not measured or controlled for by these epidemiological and quasi-experimental studies, such as the family environment and parenting, may have accounted for the associations between anemia in infancy and learning deficits later in life. IDA in infancy may also have deleterious effects on growth The available evidence shows that infants with severe IDA have impaired growth, and that correction of the anemia through supplementation can result in increased growth (Auckett et al., 1986; Briend et al., 1990; Chowang et al., 1988; Drtham et al., 1990). The etiology of the effect of iron deficiency on growth is not clear, but may involve the essential role iron plays in DNA synthesis, or alterations in eating behavior due to the malnutrition (Levitsky & Strupp, 1995) and behavioral disturbances (Walter etal., 1983,1989; Lozoff etal., 1996; Lozoff etal., 1998) associated with EDA. Walter et al. (1997) have reviewed evidence that iron deficiency may both improve and impair immune capacity and resistance to infections. Prophylactic iron has been found to increase the risk of infection in areas of the world where there is poor sanitation and disadvantaged living conditions (Murray et al., 1975a&b, 1978). The etiology of the relationship between impaired immunity and EDA is not clear, but infants with iron deficiency have been shown to have decreased levels of interleukin-2 production, which may impair cell-mediated immunity (Galan et al., 1992; Thibault et al., 1993). Evidence suggests that under usual circumstances, however, iron fortification of foods and oral iron therapy are not associated with infection, and that adequate iron status may be beneficial to immunity (Walter et al., 1997). Further research is required to clearly elucidate the relationship between iron and 13 Chapter 2. Literature Review infection. Iron deficiency has also been shown to be associated with many other functional and clinical abnormalities. These include a decrease in physical work and exercise capacity in both animals and humans (Baynes & Bothwell, 1990; Beard et al., 1990; Dodd, 1992) and impaired thermoregulation, which is thought to be mediated through the role iron plays in thyroid hormone production (Beard et al., 1990; Beard et al., 1989; Finch & Cook, 1984). A number of studies have suggested that iron deficiency may be associated with abnormalities in gastromtestinal (GI) function, contributing to such conditions as stomatitis, glossitis, hypochlorhydria, malabsorptive syndromes and GI bleeding (Baynes & Bothwell, 1990; Vyas & Chandra, 1984). Other, less specific symptoms have also been shown to result from iron deficiency including fatigue, reduced appetite, knoilonychia (spoon shaped nails) and pica (Gibson, 1990). Although it is not clear whether these conditions cause or are the result of iron deficiency, most of these systemic consequences are reversible by treatment of the iron deficiency. It is unclear how much of the association between IDA and abnormal infant behavior, growth, immunity and development is attributable to factors which are often associated with iron deficiency, such as overall poor nutrition and socio-environmental factors. Despite the lack of definitive evidence implicating iron as the causal factor, IDA in infancy is an important risk marker for poor developmental outcome (Lozoff et al., 1996; PoUitt, 1999). Iron deficiency in infancy and early childhood is clearly an important public health problem with the potential to compromise the healthy development of substantial numbers of infants and children 2.3 Iron Compounds in the Body: Chemical Properties, Distribution and Metabolic Function Although it is possible for iron to exist in oxidative states ranging from ~2 to +6, iron exists only in the ferrous (Fe2+), ferric (Fe34), and ferryl (Fe4+) states in biologic systems. This ability to exist in multiple oxidative states allows iron to transfer electrons, reversibly bind biologic ligands and, as such, participate in a number of useful biochemical reactions (Beard et al., 1996). Iron containing compounds in the body can be categorized as: 1) functional iron compounds, 2) iron transport proteins, and 3) iron storage proteins. Functional or essential iron compounds are compounds known to serve a physiologic, metabolic or enzymatic function Iron transport proteins are responsible for intracellular iron transport Iron storage proteins play a critical role in the body as regulators of iron homeostasis, and as a reserve for functional iron needs (Cook & Skikne, 1989; Dallman, 1986). The distribution 14 Chapter 2. Literature Review of iron containing compounds is summarized in Table 2.1. Table 2.1. Distribution of iron in the body. Protein Tissue site Iron content (mg) 70 Kg man1 % 10 Kg infant 2 3 % Functional compartment Hemoglobin RBC 2100-3000 67 280-320 60-70 Myoglobin Muscle 350-400 9 30-50 7 Cytochromes, other heme and iron sulfur proteins All tissues 50 1 20 4 Transport compartment Transferrin Plasma and extravascular fluid 5 <1 5 1 Storage compartment Ferritin and hemosiderin Liver, spleen, and bone marrow 0-1000 0-40 50-100 13-26 Total body iron 2505-4455 385-495 Adapted from1 Worwood M (1997), 2Oski F (1989) and Mailman P (1989). 2.3.1 Functional Iron Compounds Functional iron compounds consist mainly of heme iron proteins (e.g. Hgb, myoglobin and cytochromes) and heme containing enzymes (e.g. tryptophan pyrrolase). There are also some essential non-heme containing enzymes that either contain iron (e.g. succinic dehydrogenase) or require it as a cofactor (e.g. ribonucleotide reductase) (Dallman, 1986; Bothwell, 1995). As an integral component or essential cofactor of heme and non-heme containing enzymes, iron plays a critical role in important pathways such as DNA synthesis, mitochondrial electron transport, catecholamine metabolism, neurotransmitter levels and lipid metabolism (Larkin & Roa, 1990). A reduction in essential metabolic pathways involving iron during critical periods of development may explain why some of the effects of iron deficiency on the developing brain are not fully reversible, even when the anemia is corrected. 15 Chapter 2. Literature Review Iron serves its essential functions in the body primarily as a component of heme. Heme is a cyclic tetrapyrrole that contains iron atoms in the Fe 2 + state. Each heme molecule is bound to a polypeptide chain (globin) through a co-ordination bond of the Fe 2 + atom. It is the ability of the Fe 2 + in heme to readily associate and dissociate with oxygen, CO 2 and electrons that enables the functional iron compounds to serve their essential roles (Dallman, 1986). Circulating Hgb accounts for 65% of total body iron and functions to transport oxygen from the lungs to the tissues. Hgb, a 65-kDa oligomeric protein found in the RBC, contains 4 globin chains and 4 heme prosthetic groups. Myoglobin accounts for 5-10% of total body iron and functions to store iron for use during muscle contraction. Myoglobin is a single 17-kDa heme containing polypeptide chain found in the cytoplasm. The remainder of the functional iron present in the body, representing <1% of total body iron, is in the form of heme and non-heme containing enzymes. Cytochromes are heme containing enzymes located primarily in the mitochondria of aerobic cells, and are responsible for production of cellular energy as adenosine triphosphate (ATP) in the electron transport chain (Dallman, 1986). Ribonucleotide reductase (required for DNA synthesis and cell differentiation) and phosphoenol-pyruvate carboxykinase (required for gluconeogenesis) are examples of iron-dependent enzymes that do not contain iron but require iron as a cofactor or activator (Dallman, 1986; Sherwood et al., 1998). Iron is also incorporated into non-heme containing enzymes, including iron-sulfur proteins and metalloflavoproteins (e.g. succinic dehydrogenase), which are also involved in oxidative metabolism (Dallman, 1986). 2.3.2 Iron Transport Proteins Iron transport proteins include transferrin and lactoferrin. Transferrin is a 78-kDa single-chain (-1 globulin containing 2 binding sites for Fe3*. Although transferrin accounts for <1% of total body iron, it is the primary protein responsible for extracellular iron transport (Beard et al., 1996). Transferrin is able to bind iron and transfer it from storage to wherever it is required in the body (Cook et al., 1974). Transferrin can exist in a mono-ferric, di-ferric or apotransferrin form depending on the number of molecules of Fe3* bound. Transferrin is synthesized by the liver at a rate that is inversely proportional to the body's iron status (Sherwood et al., 1998). The primary role of transferrin is to deliver iron to the bone marrow where it is taken up for use in Hgb synthesis through receptors present on the erythropoieitc cell surface (Beard et al., 1996). In conditions of increased RBC production, such as hypoferrinemia, or when transferrin is more saturated with Fe*1", the rate of iron delivery to the cells is increased 16 Chapter 2. Literature Review (Dallman, 1986). Lactoferrin is an 80-kDa iron transport glycoprotein that is secreted from activated neutrophils and some glandular epithelial tissues. It is found in human milk, plasma and mucous secretions such as tears. It is thought to participate in the defense of the breast-fed infant against infection. Lactoferrin deprives bacteria of the iron needed for growth and donates iron to generate reactive oxygen radicals to enhance the microbicidal mechanism of phagocytes through its ability to sequester 2 atoms of Fe 3 4 per molecule. However, the role of lactoferrin in iron transport remains unclear (Beard et al., 1996). 2.3.3 Iron Storage Proteins Ferritin, the primary iron storage protein, is found in the cytosol of the reticuloendothelial cells of the liver, bone marrow and spleen. The ferritin molecule consists of a 46-kDa spherical cluster of 24 polypeptide chains that surround a colloidal core, containing variable amounts (up to 4500 atoms) of iron (Beard et al., 19%; Dallman et al., 1986). Serum ferritin values have been shown to correlate significantly with total body iron stores (Cook et al., 1974). A secondary form of storage iron, hemosiderin, is found in lysosomes of Kupffer cells in the liver. Hemosiderin is formed when the ferritin molecule becomes saturated with iron and subsequently is degraded by lysosomal proteases, thus, representing ferritin in various stages of degradation Compared to ferritin, the iron stored in hemosiderin is less chemically active and consequently not as easily mobilized from storage (Beard et al., 19%). Storage iron can vary from <5% to >30% of total body iron, depending on age, sex, weight, iron losses and previous iron nutrition This extreme intra-individual variation in storage iron can occur without apparent impairment in body function (Dallman, 1989). In infancy, the level of storage iron is relatively low and has been estimated to be approximately 10 mg/Kg (Oslo, 1989), representing about 10-20% of total body iron 2.4 Laboratory Assessment of Iron Status among Infants The natural history of iron deficiency is fairly well understood and involves 3 characteristic stages (Dallman et al., 1980; Cook et al., 1992; Bothwell et al., 1979; Suominen et al., 1998) (Figure 2.1). Each stage reflects iron status defined in relation to the amount of iron contained in the storage, transport and functional compartments. IDA in infancy is often asymptomatic and diagnosis depends on the use of appropriate laboratory 17 Chapter 2. Literature Review indices of iron status. The laboratory indices used to reflect iron status may be grouped by body iron compartment. Hgb, other RBC indices, erythrocyte protoporphyrin and transferrin receptor (TfR) concentration all reflect the functional iron compartment The tissue iron supply can be measured by serum iron, TD3C and transferrin saturation. Serum ferritin, quantitative phlebotomy and live and bone marrow tissue biopsies can be used to measure the storage iron compartment. When the dietary intake of iron is inadequate to meet requirements, storage iron is mobilized for Hgb synthesis. This continues until the iron storage compartment becomes diminished. This depletion of storage iron commonly referred to as iron deficiency or low iron stores is reflected by a progressive decrease in serum ferritin (Figure 2.1). Although indicative of risk for a deficit in functional iron and IDA, low iron stores is not, by itself, known to be associated with any functional or physiological consequences (Dallman et al., 1980). At this stage of iron depletion, all biochemical and hematological indices of iron status, except serum ferritin are normal (Figure 2.1). The 2n d stage, referred to as iron deficiency erythropoiesis (IDE), is characterised by a depletion of iron stores to the point where the levels of circulating transport iron (transferrin) are decreased. During early IDE, the only indicator is an elevated TfR concentration (Suominen et al., 1998). At this stage, due to the impairment in transport iron, the iron supplied to the erythropoietic cells is reduced. Eventually, the serum iron and the saturation of transferrin, as measured by total iron binding capacity (TTBC) become decreased. Finally in the last stage, the decrease in transport iron restricts the synthesis of Hgb and anemia develops. IDA is characterised by a declining Hgb concentration and eventually microcytic (low mean cell volume), hypochromic (low mean cell Hgb) RBCs. This is accompanied by exhausted iron stores (low serum ferritin), elevated TfR levels and decreased levels of circulating iron, as in the earlier stages of low iron stores and IDE. Individuals can be categorised according to iron status through results of blood testing to reflect the 3 distinct stages in the development of iron deficiency (Figure 2.1). 18 1 9 Chapter 2. Literature Review 2.4.1 Hemoglobin and Red Cell Indices A complete blood count (CBC) provides valuable information on RBC numbers and size and the concentration of Hgb. A CBC can provide a convenient, low cost addition to diagnostic measures for iron deficiency. A decreased mean RBC volume (MCV) and Hgb concentration (MCH) reflects a decrease in the supply of iron to the bone marrow and a decrease in Hgb synthesis. However, it can take several weeks before enough microcytic cells are released from the bone marrow to alter the MCV. The RBC distribution width (RDW) is an index of the variation in the size of the RBC that increases early in the course of iron deficiency (Dallman et al., 1996). The RDW is valuable for diagnostic and screening purposes because it is increased in IDA, but not in anemias of chronic disease, and is readily available as part of the routine CBC. However, the use of RDW for screening purposes is limited as there are no clear cut-off values that can be used because results vary according to the instrument used in the analysis (Dallman et al., 1996). Hgb is the only measure that can be used to assess the severity of the anemia, and a 10 g/L rise in Hgb after one month of a therapeutic dose of iron confirms a diagnosis of iron deficiency. Hgb concentrations have a low intra-individual variation (<5%), but show wide inter-individual variability within the physiologically normal range (Cook & Finch, 1979). Although RBC indices such as Hgb are a sensitive indicator of anemia, they lack specificity to iron deficiency and sensitivity to early deficits in functional iron (Woerner, 1988). Considering the potential consequences of IDA in infancy, screening with measures that reflect earlier stages of iron deficiency is important (Simeon & Grantham-McGregor, 1990). Developmental changes in Hgb concentration have been reviewed by Yip (1994). At birth, the mean Hgb concentration is approximately 165 g/L, and is higher than any other period in life (Saarinen & Siimes, 1978). This initial high postnatal Hgb concentration is actually a fetal adaptation to the hypoxic environment in the uterus (Dallman, 1989). After birth, Hgb levels decrease progressively until initiation of erythropoiesis at about 2 mths of age. Upon the initiation of erythropoiesis and until about 6 mths of age, there is a gradual rise in Hgb. From about 6 to 12 mths of age, Hgb tends to plateau at a mean of 115 g/L (95% Confidence Interval (CI): 105-125 g/L) (Dallman, 1989). Emond et al. (1995) found a mean Hgb concentration of 117 g/L (95% CI: 97-136) among a randomly selected sample of 8 mth old British infants. When followed into the 2n d year of life, these infants had a mean Hgb concentration of 118 (95% CI: 100-134) and 117 g/L (95% CI: 102-130) at 12 and 18 mths of age, respectively (Sherriff et al., 1999). 20 Chapter 2. Literature Review 2.4.2 Erythrocyte Protoporphyrin The concentration of erythrocyte protoporphyrin (EP) increases in iron deficiency. Its use as a parameter for defining iron status is advantageous in pediatrics because it requires a small amount of blood, and is simple to measure, rapid and reproducible. However, EP is also increased in anemia of chronic disease (Worwood, 1997). 2.4.3 Transferrin Receptor The TfR is measure of functional iron that offers a number of advantages over other laboratory measures currently used to determine iron status. Circulating levels of the soluble TfR (sTfR), a truncated form of the membrane-associated TfR, are proportional to the number of TfR on immature red cells, and thus, the rate of bone marrow erythropoiesis (Kohgo et al., 1986; Skikne et al., 1990). Expression of TfR is increased in cells where there is increased iron need and proliferation, e.g. RBC precursors and the developing placenta (Beguin et al., 1988; Baynes et al., 1994; Baynes & Cook, 1996). TfR is a transmembrane glycoprotein composed of 2 identical 95-kDa subunits linked by 2 disulfide bridges which transfers transferrin-bound iron from the circulation into the cell. Iron is taken up through receptor-mediated endocytosis of the transferrin-TfR complex, then iron is released and the remaining apo-transferrin and TfR returned to the cell surface. The number of receptors expressed on the cell surface controls the uptake of iron from the plasma into the cell (Ahluwalia, 1998). sTfR is particularly valuable in the diagnosis of IDA in conditions in which iron stores tend to be low, i.e. childhood, adolescence or pregnancy (Skikne, 1998). TfR is more sensitive than other markers of functional iron deficiency (Cook & Skikne, 1989; Skikne et al., 1990; Cook, 1999) and has a lower biological and analytical variability (Cooper & Zlotkin, 1996). Phlebotomy studies have shown that TfR values remain normal over a broad range of iron stores, and become elevated only when stores are depleted to the point that there is a deficit in tissue iron (Skikne et al., 1990; Baynes et al., 1994). Depending on the degree of the anemia, a 1.3 to 5.8-fold increase in serum/plasma TfR levels has been observed in studies of adults with iron deficiency (Kohgo et al., 1986; Thorstensen et al., 1991; Skikne et al., 1990; Ferguson et al., 1992). The measurement of sTfR in combination with Hgb is particularly valuable for the diagnosis of IDA because, unlike other laboratory indices of iron status, sTfR remains normal in anemia secondary to overt or subclinical infection or inflammation, a condition common in infancy and early childhood (Olivares et al., 1995; Ferguson et al., 1992; Punnonen et al., 1994; Pettersson et al., 1994; Thorstensen & Ramsio, 1993). 21 Chapter 2. Literature Review An elevated TfR, however, is not always specific to iron deficiency as a change in erythropoiesis will affect TfR concentrations. In hemolytic disorders, concentrations of TfR are increased 4 to 6-fold in proportion to the increase in RBC production (Huebers, 1990; Kohgo et al., 1987). Elevated TfR concentrations have been found in individuals with more severe cases of megaloblastic anemia, and with thalassemia due to ineffective erythropoieisis, in those living at higher altitudes f>1600 meters) (Allen et al., 1998), and in patients treated with erythropoietin (Ahluwalia, 1998). Mild to moderate undernutrition does not influence TfR concentrations (Kuvibidila et al., 1996), but the effect of severe protein deficiency on TfR is unknown. Low TfR concentrations (30-50% of normal) are seen in conditions involving inefficient erythropoiesis such as renal disease, aplastic anemia and post-transplantation anemia (Ahluwalia, 1998; Thorstensen & Romsio, 1993). Although several studies have involved TfR (Lonnerdal & Hernell, 1994; Virtanen et al., 1999; Yeung & Zlotkin, 1997; Anttila et al., 1997) or sTfR (Choi et al., 1999; Persson et al., 1998), information on TfR, particularly the soluble form, sTfR, in infants and children is still limited. sTfR may be particularly valuable for assessing iron status in infants because only a small volume of plasma (10 (L) is needed for the assay. Since a high sTfR is reflective of compromised iron status prior to development of anemia, it also has potential value for screening (provided less costly commercial assays become available in the future). However, information to describe the use of sTfR for defining iron status among infants in clinical settings has not yet been published. Only one study has investigated age-related differences in sTfR from the neonatal period to adulthood (Choi et al., 1999), and consistent with studies that have employed assays measuring TfR, values for infants are consistently higher than that for adults (Virtanen et al., 1999; Yeung & Zlotkin, 1997). Currently available information on TfR levels in healthy infants is inconsistent Virtanen et al. (1999) reported a mean TfR concentration of 7.8 mg/L (95% CI: 4.7-9.2) for 12 mth old infants. Yeung & Zlotkin (1997), on the other hand, found a considerably lower mean (±SD) plasma TfR concentrations of 4.4 ± 1 . 1 mg/L for 9-15 mth old infants. Similar low values have also been reported for sTfR by Choi et al. (1999) for 4-24 mth old infants (i.e. 4.5 ± 1.1 mg/L; 95% CL 2.1-6.3), and by Persson et al. (1998) for 12 mth old infants (i.e. 3.8 + 0.6 mg/L; range, 2.5-5.7). Virtanen et al. (1999) suggested that a high TfR concentration in infants and children is a response to physiologically low iron stores and, based on this, recommended age-specific reference ranges for TfR. Yeung & Zlotkin (1997), however, found no correlation between TfR concentrations and age in 9-15 mth old infants. Data on the concentrations of TfR among healthy infants over 15 mths of age have not been published, and few published data are as yet available on concentrations of the soluble form of TfR among 22 Chapter 2. Literature Review healthy infants at any age. 2.4.3 Serum Iron/TIBC and Transferrin Saturation The tissue iron supply can be reflected by several measures including serum iron, TTBC and transferrin saturation (TS). Serum iron is positively correlated with iron stores, but is decreased in the anemia of chronic disease, inflammation, and infection, and increased with iron overload. Further, the concentration of serum iron alone provides little useful information due to considerable variation from hour to hour and day to day in normal individuals (approximately 3 0 % ) . Serum iron, TTBC and TS decrease in both iron deficiency and inflammation, and thus are confounded in the same way as ferritin (Cook et al., 1993; Ferguson et al., 1992). 2.4.5 Tissue Concentrations Liver and bone marrow tissue biopsies can be used to estimate the amount of iron, either visually, using the Prussian blue reaction on tissue sections, or chemically. In the past, a bone marrow biopsy or a therapeutic trial with iron were the only means to differentiate iron deficiency from other causes of anemia Considering the invasive nature of a biopsy, however, this is an unacceptable option for confirming a diagnosis of iron deficiency in infancy. Magnetic resonance imaging (MRI) can be used to determine liver and heart iron concentrations in iron depletion, but MRI lacks the sensitivity required to distinguish minor differences in storage iron within the range of low iron stores to normal iron status (Worwood, 1997). 2.4.6 Quantitative Phlebotomy Quantitative phlebotomy is a direct way to measure iron stores, but requires removal of up to 5 0 0 mL of blood/week until anemia develops. Although quantitative phlebotomy can be used to determine iron stores for research purposes, it would not be an ethical option for research with infants, or an acceptable way to detenriine iron stores in clinical situations. 2.4.7 Serum Ferritin The utility of ferritin has been established for screening healthy individuals for a deficit in storage iron (Cook et al., 1974; Jacobs, 1977; Lipschitz et al., 1974), and for confirming iron deficiency in overtly anemic 23 Chapter 2. Literature Review patients (Ali et al., 1978). Serum ferritin is the only useful biochemical indicator of low iron stores (Baynes, 1996; Beaton et al., 1989), but a number of factors complicate its use. Although a ferritin value <12 ug/L is a highly specific indicator of iron deficiency (Ali et al, 1978), it gives no indication of the severity of the deficit in functional iron once the stores are nearly or completely exhausted (Cook & Skikne, 1989). Serum ferritin concentrations are significantly increased in iron overload and symptomatic patients with genetic hemochromotosis have values >700 (g/L (Sherwood et al., 1998). Marginal iron reserves are characteristic of infancy (Siimes et al., 1974); 5% of a large group of infants in the U.K. had serum ferritin values (16.8, 16.2 and 12.3 ug/L at 8, 12 and 18 mths of age, respectively (Sheniff et al., 1999). A wide intra-individual variability in ferritin values of about 24% has been reported (Cooper & Zlotkin, 1996), which makes concentrations close to the cut-off for low iron stores difficult to interpret. The use of ferritin as a screening or diagnostic test is also problematic because, as an acute phase reactant, ferritin may be elevated 3 to 5-fold in infants with infection, inflammation or other chronic disease, even if iron deficiency is present (Cook et al., 1993; Lipschitz et al., 1974). Iron stores at birth have been found to have a high correlation with iron stores at 6, 9 and 12 mths of age (Michaelson et al., 1995). However, it is not known whether low iron stores in the first year of life predicts risk for IDA in the 2n d year of life. Whether iron depletion in infancy results in functional abnormalities or not also remains to be determined. No single laboratory measure can adequately categorize an individual's iron status, thus a combination of measures that reflect the functional, transport and storage iron compartments must be used simultaneously to give the best picture of true iron status. Based on the available information, it seems likely that assessing sTfR in combination with serum ferritin and Hgb will facilitate characterization of iron status, from normal iron status in infancy through low iron stores and IDE to IDA. While serum ferritin is the best measure for establishing the size of the storage iron compartment, sTfR is the single best measure of the functional iron compartment (Baynes, 1996). Thus, in infancy the utility of sTfR as a diagnostic index for iron deficiency is improved when used in combination with serum ferritin. Other measures, including MCV, RDW and EP are less sensitive and predictable, and provide later indicators of the functional compartment depletion than sTfR (Baynes, 1996). Measures of Hgb provide an assessment of the severity of the iron deficiency, when evaluated along with measures of serum ferritin and sTfR. Although sTfR may be elevated due to conditions other than iron deficiency, ferritin as an acute phase protein, is within the reference interval or increased (Skikne, 1998). The measurement of both sTfR and ferritin, and calculation of the ratio of sTfR to ferritin concentration (sTfR:ferritin) is particularly valuable for assessing iron 24 Chapter 2. Literature Review status because it defines iron status over a wide range from normal iron stores to tissue iron deficiency, even in difficult situations such as rapid growth and inflammation (Cook et al., 1994; Baynes, 1996). 2.5 Iron Homeostasis and Iron Balance during Infancy A highly efficient recycling system functions to conserve body iron Iron is taken up from the circulation via transferrin and recirculated to iron-requiring tissues via specific receptors called TfR. The recycled iron is provided primarily by the breakdown of Hgb from the eiythroid marrow, or from ferritin or hemosiderin from reticuloendothelial cells or hepatocytes. The iron accumulated during a normal full-term gestation is adequate to provide the iron needed for growth and to replace iron losses for at least the first 4 mths after birth in the breast-fed infant. Iron needs in the first 4 mths after birth are met by the mobilization of iron stores, redistribution of iron, recirculation of iron from the destruction of fetal Hgb, and the contribution of breast milk iron After about 4 mths of age, however, the iron stores become depleted and the infant becomes dependent on an adequate supply of dietary iron to maintain iron balance (Dallman et al., 1980; Oski, 1989 & 1993). The exogenous and endogenous determinants of iron balance during infancy are illustrated in Figure 2.2. Unlike most other nutrients, iron balance is regulated primarily by variations in iron absorption, rather than excretion The individual's iron status and the iron supply from the diet and any iron-containing supplements are the primary determinants of the amount of iron actually absorbed by the gut The infant's endowment of iron at birth, growth rate and loss of iron, all of which vary considerably from infant to infant, determine the endogenous iron requirement during infancy. 25 Chapter 2. Literature Review 2.5.1 Iron Endowment at Birth The fetus accumulates iron throughout gestation, with the last trimester being the period of the greatest accumulation (1.7-2.0 mg iron/day) (Aggett et al., 1989). Transferrin bound iron is transferred against a concentration gradient from the maternal circulation to the fetus via the TfR on the placenta (Oski, 1989). In iron deficiency, upregulation of TfR synthesis in the placenta enables increased uptake of circulating iron This is reflected by increased cord blood serum iron, transferrin saturation and ferritin concentrations. Until recently, it was believed that maternal iron deficiency, unless severe with Hgb <85 g/L, did not compromise fetal iron accumulation (Singla et al., 1978 & 1996; Rios et al., 1975). The cross-sectional studies from which this evidence was derived, however were confounded by other factors that may have influenced the iron status of the newborn. Further the studies compared indicators of iron status only at birth, and not later in infancy. More recently, a placebo-controlled iron supplementation study repotted that iron deficiency during pregnancy does, in fact, adversely affect the infant's iron status (Preziosi et al., 1997). Although no differences were found in cord blood iron indices at birth, serum ferritin, Hgb, MCV and serum iron concentrations were significantly higher and erythrocyte protoporphyrin concentrations were lower at 3 mths in the infants whose mothers had been supplemented with iron than in those whose mothers had not (Preziosi et al., 1997). Other important determinants of the iron endowment at birth include factors such as birth weight, perinatal blood loss, an increase or decrease in the Hgb mass at birth resulting from late or early cord clamping, respectively (Grajeda et al., 1997) and the occurrence of fetal to maternal hemorrhage (Oski, 1989). Assuming a blood volume of 270 mL and a Hgb concentration of 170 g/L at birth, Oski (1989) estimated the average infant with a weight of 3 Kg has approximately 163 mg (75-80%) of iron in Hgb and a total body iron content of 214 mg, equivalent to about 75 mg iron/Kg body weight Approximately 21 mg (9%) of iron is present in myoglobin and tissue enzymes, and 30 mg (10-15%) in storage iron at birth (Heubers, 1990; Dallman et al., 1980; Oski, 1989). 2.5.2 Growth Rate The average infant triples his or her birth weight and blood volume in the first year, and as a result a relatively large amount of iron is needed for growth, i.e. 80% of the total l^uirement (Oski, 1989; Dallman, 1989). Canadian experts have estimated that about 0.34 and 0.29 mg iron/day is needed to meet the needs for iron for the 27 Chapter 2. Literature Review synthesis of Hgb and the accretion of tissue and storage iron from 5-12 mths and 1-2 years of age, respectively (Health and Welfare Canada, 1990), lower than the 0.63 mg/day estimated by Oski (1989). Although the average iron requirement for growth can be approximated, variations in growth rates are extremely large (Siimes & Salmenpera, 1989). Thus, it follows that variations in iron requirements may also be large. The peaks in the incidences of iron deficiency in infancy and childhood correspond to peaks in growth and increments in RBC mass (Owen, 1989). Among full-term infants of normal birth weight, the available evidence suggests that the more rapid the rate of weight gain during the first year, the more depleted the iron stores and the greater the risk of iron deficiency (Siimes & Salmenpera, 1989; Sherriff et al., 1999; Emond et al., 1995; Dewey et al., 1998; Michaelson et al., 1995). 2.5.3 Iron Losses The body has a limited capacity to excrete iron Basal losses of iron occur primarily through the desquamation of surface cells from the skin, GI and urinary tracts (ferritin), and small amounts of normal GI blood loss (Hgb) (Bothwell, 1995). Basal losses have been estimated to be approximately 0.04 mg/Kg/day in adults (Smith & Rios, 1974). When adjusted for the smaller body surface area, this gives a requirement for elemental iron of 0.37 mg/day from 5-12 mths (based on a weight of 9 Kg) and 0.44 mg/day from 13-24 mths (based on a weight of 11 Kg) to cover basal losses (Health and Welfare Canada, 1990). In contrast, Oski (1989) estimated that 0.13 mg/day is needed to cover basal iron losses in the first 12 mths of life. Large amounts of iron may be lost due to pathologic causes, such as episodes of diarrheal disease (Oski, 1989) and occult blood loss due to ingestion of excessive amounts of cows' milk protein prior to maturation of the GI tract (Zeigler et al., 1990), which would clearly increase iron requirements. 2.5.4 Iron Absorption The mechanism for, and mediators of the regulation of iron absorption although still not completely understood, have recently been reviewed by Beard et al. (1996) and Conrad et al. (1999). It is thought that iron absorption is regulated by mucosal cells in the upper small intestine (Bothwell, 1995). The amount of iron that is absorbed has been estimated to be about 1-2 mg/day in adults and 0.8 mg/day in infants, but actual amounts can vary about 50-fold (Dallman, 1989). Although somatic factors, such as body iron stores and rate of eryihropoiesis are the 28 Chapter 2. Literature Review main determinants of iron absorption, mtraluminal factors, such as the chemical form and oxidative state of the iron, and the presence of dietary components which inhibit and enhance iron absorption are also factors which regulate the uptake of iron into the enterocyte (Bothwell, 1995). The integrity of the mucosal surface and intestinal motility also play a role in regulating iron absorption. At physiologic intakes of iron, i.e. levels naturally occurring in food, the predominant route of absorption is by active transport that involves a series of receptors and binding proteins. At higher intakes, passive absorption via a paracellular pathway seems to play a larger role (Beard et al., 1996). It is possible that larger proportions of iron may be absorbed by passive transport in infants given high amounts of iron from iron-fortified formula or iron supplements, whereas greater proportions of iron may be absorbed by active transport in infants relying solely on the iron naturally present in food. Iron absorption occurs through 3 distinct physiological phases (Beard et al., 1996): 1) preparation of dietary heme and non-heme iron for uptake into the enterocytes in the duodenum and upper jejunum (luminal phase), 2) transport through the enterocyte (iron uptake) and 3) release from the enterocytes to plasma (iron transfer). The absorption of heme iron occurs throughout the small intestine, whereas non-heme iron is absorbed primarily in the duodenum In the upper GI tract, luminal secretions and dietary reducing agents and ligands prepare the dietary iron for absorption. In the stomach, hydrochloric acid and pepsin denature the protein to which the iron is bound and solubilize the released iron by reduction of the insoluble Fe 3 + to the more soluble ferrous Fe 2 + form. In the intestine, secretion of bicarbonate by pancreatic ducteal cells raises the pH. Although the increase in pH will theoretically decrease iron absorption, concurrent release of pancreatic proteases in the intestine chelate and solubilize the Fe 2 + to facilitate its absorption The lower gastric pH (about 5) in infancy also suggests that the efficiency of iron absorption be higher in infants than adults (Lonnerdal, 1991). Various dietary and non-dietary factors can inhibit or facilitate iron absorption. Heme iron, which is found only in animal tissues (i.e. MPF), is absorbed directly by the enterocytes via receptor-mediated endocytosis. Non-heme iron, which makes up 100% of the iron in plant-based foods, dairy products, eggs and iron-fortified products, and 60% of the iron in MPF (Monsen et al., 1978) can be present in either a Fe3* or Fe 2 + state. Non-heme iron is sequestered within the lumen and solubilized by chelators, transferred to binding proteins, and then enters the enterocyte bound to a carrier protein via receptor-mediated endocytosis. The principle pathway of absorption requires reduction of Fe3* to Fe 2 + via a reductant such as ascorbic acid. Diffusion and binding by some molecules, such as mucin, may allow small amounts of Fe3* to be absorbed directly. Fe3* absorption, however, is usually 2 9 Chapter 2. Literature Review inhibited because Fe 3 4 is easily converted to an unstable ferric hydroxide that aggregates and precipitates in the alkali environment of the intestine (Beard et al., 1996; Conrad, 1993). Extensive research over the last 3 decades using the extrinsic tag model to study the bioavailabuity of iron from single foods or meals has provided a comprehensive description of the factors that influence the absorption of heme and non-heme iron (reviewed by Lynch, 1997 and Conrad et al., 1999). Heme iron is highly bioavailable and its absorption is affected to a lesser extent by other dietary factors than for non-heme iron. Non-heme iron absorption is influenced substantially by the relative proportion of enhancers and inhibitors of iron absorption in the diet, and consequently, absorption is highly variable (Hallberg, 1974; Cook et al., 1972; Martinez-Torres & Layrisse, 1971; Turnbull et al., 1962; Conrad et al., 1966; Callender et al., 1957; Layrisse et al., 1969; Hallberg et al., 1989; Disler et al., 1975a). The currently available evidence indicates that the absorption of heme iron is enhanced only by meat, poultry and fish (MPF) (Martinez-Torres & Layrisse, 1971; Hallberg, 1981; Hallberg et al., 1992b) and inhibited by calcium (Hallberg et al., 1992a; Hallberg et al., 1991). Absorption of non-heme iron is enhanced by vitamin C (Brise & Hallberg, 1962; Hallberg et al., 1986; Rossander et al., 1979) and MPF (Martinez-Torres & Layrisse, 1971; Conrad et al., 1966; Hazell et al., 1978; Layrisse et al., 1984), and inhibited by phytate (Sandberg, 1991; Brune et al., 1992; Morris & Ellis, 1980; Reddy et al., 1996), dietary fibre (Simpson et al., 1981; Widdowson & McCance, 1942; Bjorn-Rasmussen, 1974), various polyphenols (Disler et al., 1975a&b; Gillooly et al., 1983; Disler et al., 1981; Brune et al., 1992; Macfarlane et al., 1988; Tuntawiroon et al., 1991), and calcium (Hallberg et al., 1991, Hallberg et al., 1992a,b). These factors have been shown to have a greater impact on absorption of iron in a diet that is primarily plant based and that contains no heme iron than in a mixed diet that contains heme iron (Cook et al., 1991a). The composition of the meal can also have significantly more impact on iron balance than the amount of iron, if the meal contains predominantly non-heme iron (Cook et al., 1991a). Although iron transfer initially differs for heme and non-heme iron, iron from both sources eventually enters a common iron pool in the enterocytes. Here, the enzyme heme oxygenase facilitates the release of iron from heme. The iron derived from heme sources enters a common iron pool and is then processed in the same manner as iron from non-heme sources. Iron is transferred from the common iron pool, either to ferritin in the mucosal cell, where it is eventually lost when the cells are sloughed, or to the basolateral side of the enterocyte from where it is released into the circulation After release into the circulation, TfR transfer iron from the circulation into the cell. The cell surface TfR bind Fe3+-transferrin complexes in the plasma. The Fe 3 4 is then internalized via TfR-mediated 30 Chapter 2. Literature Review endocytosis. The endosomal compartment containing the transferrin-TfR complex sheds its clathrin coat in the lower pH of the cytosol and the iron is reduced to Fe 2 + and dissociated from transferrin. The dissociated Fe 2 + is channelled into one of 3 pathways: iron-regulatory proteins, non-utilizing proteins, or storage iron. The remaining endosomal portion containing the TfR-apo-transferrin complex travels to the Golgi apparatus where it is packaged along with newly synthesized receptors and translocated to the cell surface. The higher pH of the cell surface then facilitates the release of apo-transferrin into the circulation (Beard et al., 1996). TfR are found on the cell surface of virtually all mammalian cells (Thorstensen & Romsio, 1993). The expression of TfR is regulated primarily by metabolic need and intracellular iron status and secondarily by the rate of cell proliferation (Bothwell, 1995; Beard et al., 1996). As immature RBC, eryfhroblasts and reticulocytes mature into eiythrocytes, the number of TfR on the cell surface, and thus iron uptake into the cell decreases (Beard et al., 1996). Thus, in infancy depending on the rate of growth, iron balance and the nature of the diet, the amount of iron that is actually absorbed and eventually transferred from the plasma into the cells varies considerably. 2.6 Recommendations for Iron Intake during Infancy The current Canadian Recommended Nutrient Intake (RNT) for iron is 7 mg/day for infants 5-12 mths of age and 6 mg/day for those 12-24 mths of age (Health and Welfare Canada, 1990). The U.S. Recommended Dietary Allowance (RDA) for iron is 10 mg/day for infants from 6-24 mths of age (Subcommittee on the Tenth Edition of the RDAs, 1989). These recommendations are based on theoretical estimates for iron accretion (growth and stores) and losses during infancy, with allowances for the estimated bioavailability of iron from the diet Various experts and expert groups have made different estimates for the endogenous iron requirement for the first year, which range from 0.7-0.9 mg/day (Stekel, 1984; Oski, 1989; Dallman et al., 1980; Health and Welfare Canada, 1990). The iron requirement during the first year of life for a hypothetical infant as estimated by Oski (1989) and Health and Welfare Canada (1990) is shown in Table 2.2. The amount of iron that must be supplied by the diet to meet the estimated endogenous iron need is based on the assumption that the infant consumes a mixed diet and that about 10-12.5% of the iron is absorbed (Health and Welfare Canada, 1990; Subcommittee on the Tenth Edition of the RDAs, 1989). Assuming an infant consumes 7-9 mg of iron and absorbs 10%, the endogenous iron requirement of 0.7-0.9 mg/day can be met. If the amount of iron absorbed from the diet is insufficient to meet the tissue needs, then the iron status 31 Chapter 2. Literature Review of the infant will become compromised, leading to decreased iron transport, Hgb synthesis and eventually, IDA. The dietary recommendation for iron is based on many approximations and assumptions concerning the iron endowment at birth, rate of weight gain, iron losses and the iron content and composition of the diet throughout infancy. The absorption of dietary iron varies from a few percent to 50%, depending on the composition of the meal and iron status of the individual. Considerable variation from infant to infant exists in all of these variables, placing some infants at risk of iron deficiency if intakes are inadequate to meet their requirements. 32 Chapter 2. Literature Review Table 2.2. Changes in body iron during infancy during the first year of life in a hypothetical infant and estimated requirements for endogenous and dietary iron. 1 A 8 e Estimated iron Endowment at birth Estimated body iron atone year of age Daily iron requirement Health and Oski, 1989 Welfare Canada, 1990 Weight (Kg) 3 10 Hemoglobin (g/100 mL) 17 11 Blood volume (mL/Kg) 90 75 Total blood volume (mL) 270 750 Total body hemoglobin (g) 46 82 Hemoglobin iron (mg) 163 280 Tissue iron (7 mg/Kg) 21 70 Storage iron (10 mg/Kg) 30 100 Total body iron (mg) 214 450 0.65 0.34 Totally yearly iron losses (0.13 mg/day) (ug/day) — 47 0.13 0.37 Exogenous iron requirement (mg) — 283 0.78 0.71 Daily dietary iron requirement (mg)2 — — 8 7 'Adapted from Oski, 1989. 2Assuming an absorption of 10% of the dietary iron. 33 Chapter 2. Literature Review 2.7 Sources, Amount and Bioavailability Dietary Iron in Infancy The amount and bioavailability of iron in the diet varies considerably, depending on the foods consumed. Feeding practices, therefore, play a critical role in the development of poor iron status, i.e. iron deficiency anemia or low iron stores, during infancy. Cook & Bothwell (1984) have described the nature of dietary iron during infancy in relation to 3 overlapping periods (Figure 2.3). According to current recommendations (Canadian Pediatric Society (CPS) et al, 1998; American Academy of Pediatrics Committee on Nutrition (AAP-CON), 1999), breast milk and/or infant formula should be the sole food until 4 to 6 mths of age. Between 4 and 6 mths and continuing throughout the first year, complementary foods, starting with iron-fortified infant cereals, then vegetables, fruits, and finally MPF and alternatives are recommended. It is also recommended that breast-feeding or feeding with a commercial iron-fortified infant formula continue after the introduction of solid foods, and that iron-fortified foods continue beyond the first year to provide sufficient iron. Breast-feeding is recommended until up to 2 years of age, or beyond. For the infant who is not breast-fed, a commercial iron-fortified infant formula is recommended until 9-12 mths of age (CPS et al., 1998; AAP-CON, 1999). The process of weaning commences at 4-6 mths, with an increasing dependence on solid foods during the latter part of infancy and into the 2n d year. By one year of age, the ingestion of a variety of foods from the different food groups of Canada's Food Guide to Healthy Eating is recommended. Full-term gestation infants are able to draw upon the storage iron laid down during gestation for at least the first 4 mths after birth, and as a result, the amount and bioavailability of dietary iron is not so important to iron balance during this time. From 4-6 mths of age to the 2n d year of life is a critical period for iron balance because these reserves become depleted, and the major sources of dietary iron are undergoing an enormous change from solely breast milk or infant formula, to a diet that includes an increasing variety and amount of complementary foods. 34 Chapter 2 . Literature Review Age (mths) Figure 23. Schematic illustration of the major sources of dietary iron, and inhibitors and enhancers of iron absorption, shown with reference to the recommended patterns of food consumption during infancy.1 'Adapted from: Cook & Bothwell, pg. 119. In Sketel A. (ed) Iron Nutrition in Infancy and Childhood. Raven Press, New York, 1984; Canadian Pediatric Society, Dietitians of Canada, Health Canada, 1998. Arrows indicate the recommended ages of introduction (CPS et al., 1998). 2.7.1 Human Milk The iron content of human milk is relatively low and quite variable, depending on the stage of lactation. The amounts of iron in human milk range from 0.5-1.0 mg/L iron early postpartum to approximately 0.3-0.4 mg/L after the first few mths (Bates & Prentice, 1994; Fomon et al., 1993). The iron content of human milk is neither related to maternal iron intake nor influenced by iron supplementation (Bates & Prentice, 1994). Although the iron content of human milk is low and in the ferric form, the absorption of iron from an exclusive breast-milk diet is relatively high, up to approximately 50% (Saarinen et al., 1977; MacMillan et al., 1976). Recently, the iron erythrocyte incorporation method has been used to estimate iron absorption from human milk These studies found similar rates of iron absorption of about 12% among 8 breast-fed infants aged 2-10 mths (Davidson et al., 1994a) and 14 breast-fed infants aged 5-7 mths (Abrams et al., 1997). The variability in the iron absorption from human milk, however, is wide, with a range of 3.4-37.4% (Davidson et al., 1994a). Assuming the infant is exclusively 35 Chapter 2. Literature Review breast-fed, an intake of 750 mL human milk/day would provide 0.26 mg, based on a human milk iron content of 0.35 mg/L. Assuming a maximum bioavailability of 50% the amount of iron absorbed would be 0.13 mg/day, although actual intakes and amounts absorbed by infants may be considerably lower than this. The intake of iron from human milk is clearly much lower than the requirement of 0.7 mg/day that has been estimated for infants 5-12 mths of age (Health and Welfare Canada, 1990). Further, not only is the amount of iron in human milk insufficient to meet the needs of infants over 5 mths of age, feeding solid foods near the time of breast-feeding decreases the absorption of iron from human milk (Oski & Landaw, 1980). 2.7.2 Infant Formulas and Cows' Mi lk The iron content of infant formulas varies considerably, and the bioavailability of the iron tends to be lower than that in human milk. Unfortified (low iron) cow's milk-based infant formulas in Canada contain approximately 1.5-3 mg/L iron in the form of Fe34, that is naturally present in cows' milk. The absorption of iron from low iron formulas is about 10% (Stekel et al., 1986; Saarincn & Siimes, 1977). Iron-fortified infant formulas typically contain 7-13 mg iron/L. The iron in iron-fortified infant formulas is in the form of Fe^ SO*2"* ferrous sulfate, a form that is more readily absorbed when compared with other forms of iron used to fortify foods, such as elemental iron powders and phosphate compounds. The absorption of iron from iron-fortified infant formulas is generally inversely proportional to the iron content of the formula (Macmillan et al., 1977; Fomon et al., 1997). Using the erythrocyte incorporation method to estimate iron absorption, Fomon et al. (1997) found that infants absorbed 3.5% and 2.6% of the iron from formulas containing 8 and 12 mg iron/L, respectively. Iron-fortified soy protein formulas contain higher amounts of iron (12-13 mg) because it is thought that the iron from these formulas is more poorly absorbed (about 2-3%) than from cows' milk protein formula (Hertrampf et al., 1986; Brennan et al., 1989; Gillooly et al., 1984). Values for the absorption of iron from soy protein formulas, however, have been extrapolated from studies in adults. Iron absorption, based on the 59Fe eiythrocyte incorporation method by infants, however, is thought to be about 2-fold higher than by adults (Lonnerdal, 1990). Rios et al. (1975) and Davidson et al. (1994b) found 4-6% iron absorption from soy protein formulas in infants 3-7 mths of age. Unmodified cows' milk contains approximately 0.5 mg iron/L, with an absorption of about 10% (Saarinen & Siimes, 1979). Although the mechanism is not entirely understood, the relatively higher absorption of iron from human milk compared with cows' milk, or soy and cows' protein infant formulas is thought to be due to the lower calcium 36 Chapter 2. Literature Review and protein content and the presence of lactose and lactoferrin in human milk, which are thought to facilitate iron absorption (LSnnerdal, 1990; Lynch & Hurrell, 1990; Hallberg et al., 1992a). 2.7.3 Complementary Foods Commercial infant cereals, and in older infants other iron-fortified cereals and MPF, are the major sources of iron from complementary foods throughout the weaning period (Lynch & Hurrell, 1990). With the exception of MPF and non-fortified products, complementary foods tend to be poor sources of iron (Zeigler & Fomon, 1996). A summary of the food and nutrient intakes from 4 national surveys in the U.S. has shown that solid foods provide over 95% of the iron in the diets of infants fed unmodified cows' milk and low iron infant formulas during the 2n d 6 mths of life (Ernst et al., 1990). The amounts and sources of iron, or inhibitors and enhancers of iron absorption from solid foods in the diet of the breast-fed infant have not been determined. Infant cereal is the first and most commonly used complementary food (Zeigler & Fomon, 1996; Skinner et al, 1997), and is recommended as a key source of iron for late infancy (CPS Nutrition Committee, 1991; CPS et al., 1998). Infant cereals are fortified at a level of 30 and 45 mg/100 g dry cereal in Canada (Health and Welfare Canada, 1997) and the U.S. (Zeigler & Fomon, 1996), respectively. The iron in iron-fortified infant cereals has a bioavailabiUty of only 3-4% (Fomon, 1987) because these foods contain inhibitors of iron absorption (fibre and phytate), and the iron is in the form of poorly absorbed electrolytic iron powder. Nonetheless, iron-fortified infant cereals provide most of the dietary iron for infants 7 to 10 mths of age fed cows' milk or low iron formulas (Ernst et al., 1990). Further, iron-fortified infant cereals provide >50% of the iron in the diets of infants fed iron-fortified formula at 11 to 12 mths of age (Ernst et al., 1990). Although introduction of iron-fortified infant cereals at 4-6 mths and continued feeding to at least one year of age has been recommended as the primary strategy for prevention of IDA among breast-fed infants in Canada since 1979 (CPS Nutrition Committee, 1979 & 1991; CPS et al., 1998), the practicality of this recommendation may be a problem for some infants. Yeung et al. (1981) found that although the majority of infants were introduced to iron-fortified infant cereals by the recommended age of 4-6 mths, the cereals were discontinued within one to 3 mths for a large proportion of the infants. Further, data from Walter et al. (1993) suggests that breast-fed infants have lower intakes of iron-fortified infant cereals than formula-fed infants. In a study of the feeding practices of Chinese infants 12-18 mths of age living in Northeast Edmonton, infant cereal was considered by some mothers to be a "hot" or yang food that causes constipation and was often avoided (unpublished, 37 Chapter 2. Literature Review Hui, 1997). Rather, congee, traditional rice gruel very popular in Chinese diets, was fed to infants as the first complementary food instead of commercial iron-fortified infant cereals (Leung & Davis, 1994; Li, 1985). The methods for preparing congee vary considerably. Although MPF is sometimes added to the congee, the MPF is usually not fed to infants. Thus, congee tends to have a low iron content and bioavailability (Dallman & Siimes, 1979a; Hallberg et al., 1977; Hsia & Yeung, 1976). Although a recent study in Vancouver suggests that most infants are introduced to iron-fortified cereals by the recommended 4-6 mths of age (Williams et al., 1996), data on the quantity and/or the duration of infant cereal feeding throughout the 2nd 6 mths of life was not collected. In addition to having a low iron content (approximately 0.4 mg/100 g), fruits and vegetables contain non-heme iron, and have been shown to interfere with the absorption of iron from breast milk in adults (Oski & Landaw, 1980). However, Walravens et al. (1989) reported that because of the quantity consumed, fruits and vegetables actually contributed 12% and 8% of total iron intake, respectively, among infants from low-income families in Denver. Legumes, eggs and dairy products were found to contribute 5% 5% and 4% respectively, to the total iron intake. Although the non-heme iron in these foods may contribute substantial amounts to total iron intakes and their vitamin C contents enhance non-heme iron absorption; they contain relatively high amounts of inhibitors of iron absorption, such as dietary fibre and phytates. An adequate intake of iron from solid foods is particularly important for infants who do not receive an iron-fortified infant formula or supplemental iron Table 2.3 shows the estimated amount of iron from solid foods that needs to be ingested to meet the endogenous iron requirements of infants 6-24 mths of age based on the primary milk feeding. As shown, the amount of iron that needs to be supplied by solid foods varies considerably depending on the amount of iron provided by the primary milk feedings. There are no recent published data on the intake and duration of feeding of iron-fortified infant cereals, other complementary foods, or the overall composition of the weaning diet for Canadian infants during the first to 2nd year of life. 38 Chapter 2. Literature Review Table 2.3. Estimated amount of iron from solid foods needed to meet the endogenous iron requirement of infants 6-24 mths of age in relation to type of primary milk feeding. Primary milk feeding Estimated maximum iron provided by milk1 Endogenous requirement not met by primary milk feeding Estimated iron intake from solid food needed to meet endogenous requirement (mg/day) Iron content (mg/L) Estimated absorption _ i % L 1 2 W 10° Estimated iron absorbed 5-12 mths2 13-24 mths3 Breast milk Low iron Formula Cows' milk Iron fortified Formula Soy protein-based formula 0.35 1.5-3.0 0.5 7-13 12-13 10' 4-6y 0.03-0.13 0.15-0.30 0.04 0.21-0.39 0.23-0.58 0.57-0.67 0.40-0.55 0.66 0.31-0.49 0.12-0.47 5.7-6.7 4.0- 5.5 6.6 3.1- 4.9 1.2- 4.7 4.6-5.4 3.2-4.4 5.3 2.5-3.9 1.0-2.2 1 Assuming a maximum intake of 750 mL/day 2 Assuming a mixed diet containing heme iron sources with an absorption of approximately 10%; based on an endogenous iron requirement of 0.7 mg/day (Health and Welfare Canada, 1990). 3 Assuming a mixed diet containing heme iron sources with an absorption of approximately 12.5%; based on an endogenous iron requirement of 0.7 mg/day (Health and Welfare Canada, 1990). "Davidson et al., 1994a; Abrams et al., 1997. 5Saarinen et al., 1977; MacMillan et al., 1976. 6Skekel et al., 1986; Saarinen & Siimes, 1977. 'Saarinen & Siimes, 1979. 8Fomon etal., 1997. ^os et al., 1975; Davidson et al., 1994b. 2.8 Adequacy of Iron Intakes in Infancy Several studies have shown that heme iron accounts for only a small proportion (about 6%) of the total iron intake of infants and young children (Gibson et al., 1988; Raper et al., 1984; Preziosi et al., 1994; Skinner at al., 1997). Only one study has reported the dietary intake of iron among infants in China (Chen et al., 1992), and there is no published data on the intake of heme and non-heme iron intakes in Chinese infants. Data from the recent Chinese National Nutrition Survey suggest that heme iron intakes of Chinese infants may be low; although total dietary iron intakes among the study population were high, heme iron only accounted for about 3% of total iron intakes, with lower intakes in rural than urban areas (Du et al., 2000). Despite an increase in solid food intake from the start of weaning into the 2nd year of life, a downward trend in iron intake occurs (Zeigler & Fomon, 1996; Yeung et al., 1981; Richmond et al., 1993; Brault-Debuc et al., 1983). 39 Chapter 2. Literature Review High within- and between-subject variation, particularly from 12 to 24 mths of age has been found for iron and vitamin C intakes of Asian children for whom weighed intake data was kept (Harbottle & Duggan, 1994). Tables 2.4-2.6 provide a summary of studies that have examined iron intakes in infancy and early childhood. Studies in the U.S. have generally shown that the intake of iron by infants has increased over the last 3 or 4 decades (Zeigler & Fomon, 1996). Despite this, studies in the U.S. (Richmond et al, 1993; Johnson et al., 1994; Zive et al., 1995) as well as Western Europe (Harbottle & Duggan, 1994; Calvo & Gnazzo, 1990; Murphy et al., 1992) have identified a high prevalence of inadequate iron intakes during infancy and early childhood. Levels of food iron fortification vary dramatically from country and country, thus comparison and extrapolation of data from other countries to Canada difficult. The levels of iron fortification of foods such as breads and cereals, infant foods, and soy protein-based products in Canada are generally considerably lower than in the U.S., and thus it would be expected that iron intakes would be lower. Few Canadian studies, however, have determined the intake and sources of iron in the diet throughout infancy (Greene-Finestone et al., 1991; Brault-Debuc et al., 1983) and early childhood (Gibson et al., 1988), and no studies conducted since 1985 have been published. The data collected by Greene-Finestone et al. (1991) in 1985 on infants 6-18 mths of age hving in Ottawa-Carlton, and by Brault-Debuc et al. (1983) in 1975-1979 on French Canadian infants living in Montreal, show that despite median iron intakes that met or exceeded the RNI, about 20-35% of the infants had iron intakes below the RNI. Similarly, Yeung et al. (1981) found that as many as 35% of 6 mth olds, and 37% of 10 mth olds in Toronto had iron intakes below the RNI, with a notable increase in the proportion of infants that did not meet the RNI for iron with increasing age. The degree of inadequacy of iron intakes in these studies has not been reported, although Brault-Debuc reported that about 10% of infants had iron intakes <77% of the RNI, an amount that is thought to be associated with a high risk of deficiency. The main reason for the declining iron intake in relation to the RNI in the infants studied in Toronto was the early withdrawal of infant cereals from the diet (Yeung et al., 1981). No data on the intakes of heme and non-heme iron, or other dietary factors influencing iron absorption among Canadian infants, or on the iron intakes or composition of the weaning diet among infants from Chinese ancestries in North America have been published. Further, the data on the intake of iron reported by Yeung et al. (1981), Greene-Finestone et al. (1991) and Brault-Debuc et al. (1983) is about 20 years old. Thus, there is currently no contemporary data on the iron intakes or food sources among infants in Canada. 40 (73 >\ u C o ft 55 E £ s s cs H cs b^ 3 b ° O = Vi c w E 0> "O 3 a. o a. T3 3 * I H CO o z pi z (N o T3 I H Pi-z Pi z \t o\ o ™ o\ -a- o r~-io u l s 1 ~—' 1 CN ' ^ ' CN SO O I t T3 cd C " ca Pi OS ~ 6 & o S c3 Pi CN <N — OS — i i i —. -H oo OS O <*> r ? ,5 2 . >-} PH 00 ^ \B W C VI « <D 32 o u 3 > Q 3 % '£fc Z & 0) 00 3 ^ -S "3 I < U 05 o Pi V N «J IT) C 90 •a os cs r oo i OS V) O PH Z < Q oo D «8 in '"C o CN Pi z Pi z fsT so" os CN oo p fl © CN CN o od •<t O so C- CN CN os OS —; •5f OS fl OS p OS t< so I t"^  1 O 1 fl i H H I • •—1 PH RT PH CN p L , PH ' PH 1 PH i A S A S A s FF fc" u fc ° PH U PH u PH FF u (NIF/ (NIF/ (NIF/ (NIF F(NI PH PH PH FF PH PH PH PH FF cn ^ sd 0 0 I PH i PH ~ & S o ^ u o H in H M CN fl CN OO H m O o -o c. Pi 00 ^ °^ ZH o 00 t SO OS T3 >s c « * 3 H ^ ^ OO K -2 Z •2 e z •a * ^ Z w CJ -t^  OS S © j. 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OS Os t~-C o , eu r o J ; {J 00 a 3 2 u 3 _f a, 3 VO i Z CO c O JX 3 o " oo B 00 © Os X i „ co o C J CN Z ci Z o ro o " +1 ^ vo of o CN ro o o oo I VO oo Os e o cd U i cd cd C O O i H CU C J U fa 44 Chapter 2. Literature Review 2.9 Prevalence of Iron Deficiency in Relation to Risk Factors Iron deficiency is the most common micronutrient deficiency among infants worldwide (United Nations, 1989; deMaeyer et al., 1985; Scrimshaw, 1991). It has been estimated that 25% of infants worldwide, and up to 10-12% of infants in developed countries have IDA (deMaeyer et al., 1985; Stevens, 1991), with an even higher prevalence among certain subgroups of the population. A summary of the prevalence of IDA and low iron stores among infants in developed countries is shown in Table 2.7. The prevalence of iron deficiency in infancy varies considerably depending on socio-demographic factors (such as age, ethnic background and socio-economic status SES), physiologic factors (such as low birth weight, premature delivery, chronic hypoxia, perinatal bleeding, a low Hgb concentration at birth and frequent infections), and dietary factors (such as feeding history, and the intake of iron and other dietary factors influencing iron absorption). Table 2.7. Summary of data reported on the prevalence of iron deficiency anemia and low iron stores among infants in developed countries. . . i » i / n / v i Authors, yr of publication Country and region Prevalence (%)1 J Iron Low iron deficiency stores anemia Canada Halifax, Toronto, Edmonton 33 Zlotkin et la. 1996 and Montreal 4 Vancouver Chinese 4 12 Innis etal., 1997 Caucasian 8 25 Innis etal., 1997 Montreal Low SES 25 37 Lehmannetal., 1992 Chinese 12 — Chan-Yip & Gray-Donald, 1987 Aboriginal Moffatt et al., 1991; Cruz et al., 32-50 — 1990; Whalen et al., 1997; Sawchuk et al., 1996; Willow et al., 2000 as National sample (NHANESffl) 3 — Looker et al., 1997 WIC participants 35 — Gupta etal., 1999 Europe National sample (Euro- 10 25 Male etal., 1995 Growth project) SES, socio-economic status; NHANES HI National Health and Examination Survey HI; WIC, Special Supplemental Food Program for Women, Infants and Children. 'The criteria used to define iron deficiency anemia and low iron stores varies from study to study. 45 Chapter 2. Literature Review There has been an overall decline in the prevalence of IDA over the past 30 years among infants in the U.S. (Looker et al., 1997; Dallman, 1990; Yip et al., 1987a&b). The prevalence of IDA declined from 7 to 3% from 1976 to 1985 among low-income families, and from 6 to 3% from 1968 to 1973 among middle-income families from 6 states in the U.S.. The prevalence of IDA in the U.S. in 1993 was at or below 3% for children aged 1-5 (U.S. Preventive Services Task Force, 1996; Looker et al., 1997). The apparent decrease in the prevalence of IDA in the U.S. has been attributed to increases in the incidence and duration of breast-feeding and feeding with non-fortified rather than unfortified infant formulas and cereals. These changes were in part due to the Special Supplemental Food Program for Women, Infants and Children (WIC) program in the U.S. (Yip et al., 1987a&b; Miller at al., 1985; Vazquez-Seoane et al., 1985). A report by Kwiatkowski et al. (1999) showing that 55 children between the ages of one and 3 years presented to the Children's Hospital of Philadelphia with severe IDA (Hgb ^60 g/L) due to nutritional reasons suggested that IDA may be a more substantial problem among certain groups. A recent report by Gupta et al. (1999) also suggested that aggregate programmatic data might not accurately represent the prevalence of IDA among particularly high-risk groups, such as WIC recipients. The prevalence of anemia (defined as a Hgb <112 g/L) among children 6 mths to 5 years of age born to adolescent mothers of low SES was 35%, despite the mothers having received WIC services aimed at prevention of IDA and a history of use of iron-fortified infant formula which approached 100% (Gupta et al., 1999). National programs similar to WIC do not exist in Canada, and studies suggest that IDA may also be a substantial problem among certain subgroups in Canada (Lehmann et al., 1992; Chan-Yip & Gray-Donald, 1987; Innis et al., 1997; Moffatt et al., 1991; Cruz et al., 1990; Whalen et al., 1997; Sawchuk et al., 1996; Male et al., 1995). National data are not available on the prevalence of iron deficiency in Canada. Data from the Euro-Growth project, however, found that the prevalence of EDA among 12 mth old infants from different regions of Europe, which also have no programs similar to WIC, was about 10% (Male et al., 1995). Recent data from major urban centres in Canada indicated that 4.3% of 8 to 15 mth old infants in Halifax, Toronto, Edmonton and Montreal (Zlotkin et al., 1996), and 7% of 9 mth old infants in Vancouver had EDA (Innis et al., 1997). Of concern, infants from certain subgroups in Canada appear to be more vulnerable to EDA, with prevalence rates of 24% among infants from disadvantaged families (Lehmann et al., 1992), 11.4-16.5% among infants from Chinese ancestries in Montreal (Chan-Yip & Gray-Donald, 1987) and 32-50% among infants from aboriginal communities across Canada (Moffatt et al., 1991; Cruz et al., 1990; Whalen et al., 1997; Sawchuk et al., 1996; Willows et al., 2000). The risk of EDA is 46 Chapter 2. Literature Review also increased among infants who are exclusively breast-fed beyond 3-6 mths (Innis et al., 1997; Siimes & Salmenpera, 1984; Calvo et al., 1992; Pizarro et al., 1991). Breast-feeding has become more common among Canadian women (Williams et al., 1996, Health and Welfare Canada, 1991; McNally et al., 1985). In Vancouver, 15% of 9 mth old infants who were breast-fed for 8 mths had IDA (Innis et al., 1997). Moreover, more Canadian families are living in disadvantaged circumstances (Canadian Council on Social Development, 1997; Mclntyre et al., 1998). Modifiable dietary factors have been shown to be important predictors of the risk for IDA in infancy (Pizarro et al., 1991; Boutry & Needlman, 1996). Dietary factors can, therefore, be targeted in initiatives aimed at preventing EDA. Socio-demographic markers of risk for iron deficiency such as age, ethnicity and SES can be used to identify infants at risk for feeding practices and dietary intakes associated with iron deficiency. Although numerous studies have identified socio-demographic and dietary factors that can be targeted in strategies aimed at prevention of EDA, a complete understanding of these factors is lacking for infants at risk for EDA in Vancouver. 2.9.1 Infant Age as a Predictor of Risk for Iron Deficiency The risk of iron deficiency between the ages of 6 and 24 mths is high, and data from the U.S. (Sargent et al., 1996; Looker et al., 1997) and Europe (Hercberg et al., 1987) suggest a decrease in the prevalence of EDA occurs after the 2n d year of life. Whether the risk of EDA differs by age between 6 and 24 mths is less clear. Although few longitudinal studies have been done (Brault-Debuc et al., 1983), cross-sectional studies in Canada (Greene-Finestone et al., 1991; Chan-Yip & Gray-Donald, 1987) have suggested that the prevalence of EDA may increase from the first to the 2n d year of life. The prevalence of EDA was higher among infants from higher SES backgrounds in Ottawa-Carlton at 18 mths (10.5%) compared with 6 mths of age (3.5%) (Greene-Finestone et al., 1991). Similarly, Chan-Yip & Gray-Donald (1987) found that me prevalence of EDA was 11.4% at 6-12 mths and 16.5% at 19-36 mths of age among infants from Chinese ancestries in Montreal. In a study with 9 mth old infants in Vancouver, 8% of Caucasian but only 4% of Chinese infants had EDA and 25% of the Caucasian and 12% of the Chinese infants had low iron stores (Innis et al 1997). Consistent with this and national data from 1970-72 (Vallberg et al., 1976), recent data from 4 major cities in Canada, Zlotkin et al. (1996) reported that a high prevalence of low iron stores affecting 25-30% of infants at about one year of age. Whether low iron stores are naturally corrected in the 2n d year of life as the variety and amounts of solid foods increase, or whether low iron stores persist or lead to 47 Chapter 2. Literature Review EDA is not clear. 2.9.2 Ethnic Background as a Predictor of Risk for Iron Deficiency Studies reporting data on the prevalence of EDA and low iron stores in infants from Caucasian and Chinese ancestries are summarized in Tables 2.8 and 2.9, respectively. Based on the findings of Chan-Yip & Gray-Donald (1987) that 11.4% of Chinese infants in Montreal at 6-12 mths and 16.5% at 19-36 mths of age had EDA, the Canadian Task Force for Periodic Health Examination (1994) categorized infants from Chinese ancestries as a high risk group for EDA. Severe EDA (Hgb (60 g/L) has also been reported to be common in children from Southeast Asian ancestries in Philadelphia at one to 3 years of age (Kwiatkowski et al 1999). The only other Canadian study that examined the prevalence of EDA among Chinese infants found only 4% EDA at 9 mths of age (Innis et al., 1997). Consistent with the latter data for Chinese infants in Vancouver, Sargent et al. (1996) found low rates of iron deficiency among children 6 mths to 5 years in communities in Massachusetts with either more than 2% or less than 1% Chinese in the population (OR 1.01, CI 0.96-1.07). EDA has also been found to be uncommon among 18 mth old infants in Hong Kong, affecting only 2% of the infants (Chiou et al., 1990), although rates of EDA in rural China are reported to be much higher than in urban areas (Ge, 1995). The low prevalence of EDA among the Chinese infants in Vancouver (Innis et al., 1997) and in Hong Kong (Chiou et al., 1990) was attributed to a high prevalence of feeding with iron-fortified infant formula Although the high prevalence of feeding with iron-fortified infant formula among Chinese infants in Vancouver protects them from EDA in the first year of life, it is possible that the latter reliance on low iron complementary foods, such as congee, and high intakes of cows' milk (Leung & Davis, 1994; Li, 1985; Chan-Yip & Gray-Donald, 1987; Hui, 1997; Kwiatkowski et al 1999; Guldan et al., 1993) places them at risk for EDA in the 2n d year of life. Alternatively, accumulation of high iron stores during the first year of life may protect these infants from later low dietary iron intakes. The Canadian Task Force for Periodic Health Examination (1994) did not define infants from Caucasian ancestries as a group at high risk for EDA. This was based on the data of Greene-Finestone et al. (1991) and Brault-Dubuc et al. (1983) that showed a low prevalence of <5% EDA among Caucasian infants from families of high SES in Ottawa-Carlton and Montreal, respectively. More recent studies, however, found 8% EDA and 25% low iron stores among 9 mth old Caucasian infants in Vancouver (Innis et al., 1997), and 25% EDA and 37% low iron stores among 10-14 mth old infants from disadvantaged families of predominandy Caucasian ancestries in Montreal 48 Chapter 2. Literature Review (Lehmann et al., 1992). The high prevalence of IDA among Caucasian infants in Vancouver was associated with a high prevalence of breast-feeding for >3 mths. Although the high prevalence of breast-feeding may place Caucasian infants at risk for IDA in the first year of life, it is possible that the subsequent introduction of a variety of iron-fortified and heme iron containing complementary foods corrects and decreases the risk of IDA in the 2n d year of life. 49 i SJ W St CJ S .2 a a CS § a o © a s 1 at 1 8J i>> CJ a V CJ a 2 s e OB 0) o at a a s CO 00* cs 3 es H a O s CN alen alen Pre ron cienc emia ron cienc emia CN s o «3 CS 0 on I *-* a 2 hH h i .2 at •c •*•» •c .a. o O a s a o 3 2 © a 2 i u s *> at c a OJ V •o a a " 2 a o CJ 13 5. 3 u o *—( VI •a '6 P H -r ™ v a v • cS CN * 14 oo 00 t o u CO I S CN • o os $ 00 CN ill 1 a a 0 0 • * SO S CN CO o 0 i 01 a S O a <? O S C s ) 5 t "E o P H ii! m V oo tJ PQ S O f > T3 o e o o a H CN SO 'E * P H C N r-W CO ^ £ ^ O OH a e C I 00 OS I u OS at ^ xf.2 a o V 00 B g 5 co VI I P H •5 2 14 O CN cn 00 f - H I SO <u CO T 3 CO •a . - 2 00 O S a a C N oj-a CN SO r-cn cs o VI yj | 8 2 o VI x. > 0) P H 00 CN • o o O S I O S 00 O S •a 1 3 ® s 3 * 50 <u 3 O s § on oj s I on | 5 on a o 2 e a 2 is e a CJ a .Si '<j c oi •a a © a o o S a a 00* c4 3 H a CU 13 P H a © CJ C u I a s M <s ett •c CU •c a I 1 5«N> a -2 1-0 S T 3 o IS a £ it 3 c CJ •= c a CU CU a s a © Cu © •a o p a © ett o - "-S u et) © ej •« "S 3 >- s r o r o T t ' •a •§> •c =t-tin ° 2 \ / l vi .3 00 T f VO <tH c tin a o o o o •If-I I 1 & S ? f (2 ft'2 §, o H a © OS ON CN O oo' © 3 •—i o VI V I - .S g .j| •^ « ^ x> V I r o .tt -e o a | r o T f r o ON ON CD 1 U -si . 2 t -B ON 3 5 51 a <u H P H e o • pH i a s HH tS at •c « •c u a 2 ? a-*> at c S •o a a " O < e a o 3 s co a. o P H >>> M SO a o a s s u C v •o a S o - "5 1m at o w -O «R -3 •< s-» a. a 2 ^ vi 5 • S i (2 cn •a O VI 2 ^ 2 vi g J •B 11 '6 © i A O u S ' v i . g 141 ON ON o u •d .2 r-~ a o\ 6 00 T t ON >—i ON 1 S oo 'V CS - H ON H • • cn NO VO* r-VI "E . 5 NO cn NO cn • NO cn cs 00 1 1—I I VO •Mi cs ^ L J CN cn • 1 1 NO f S t C S 5 £; cs ON 00 •a f o S « o s ON oo 60 a o ev 00 Tl Tt cs cn i i 11 " * NO cs cn .—i NO ^ V7 VI S ?s V : i <*> R y £ S 2 > o 1, •a VI o NO NO cn i NO ON 00 11 2* 3 ON © T-C *s -u a 52 Chapter 2. Literature Review 2.9.3 Socio-economic Status as a Predictor of Risk for Iron Deficiency Studies in both Canada (Lehmann et al., 1992; Greene-Finestone et al., 1991) and the U.S. (Yip et al., 1992; Sargent et al., 1996) have suggested that a low SES family background places an infant at risk for EDA. Among 320 infants 6 to 18 mths of age in Ottawa-Carlton, only 2-3% from high and middle SES groups had EDA compared with 8.2% from the low SES group (Greene-Finestone et al., 1991). Infants 10 to 14 mths of age from disadvantaged families in Montreal have also been found to have a high prevalence of EDA and low iron stores of 24 and 37% respectively (Lehmann et al., 1992), while EDA affected <5% of infants 3-36 mths of age from upper-middle class families (Brault-Debuc et al., 1983). The prevalence of iron deficiency in the NHANES I study in the U.S. in 1968-73 was 21% among 12 to 36 mths old infants from low-income families but only 7% among infants from higher-income families (Dallman et al., 1984). Similarly, data from the Centre for Disease Control (CDC) Pediatric Nutrition Surveillance System (PedNSS) in 1980-91 indicated that the prevalence of EDA among low-income children in the U.S. was 20-30% (Yip et al., 1992). However, the high prevalence of EDA reported in these U.S. studies may in part have reflected preferential enrolment and retention of anemic children by public health nutrition programs. Indeed, the national prevalence of EDA reported by Yip et al. (1992) for young children in the U.S. during this same period was 5%. The higher prevalence of EDA among infants from low compared with high SES family backgrounds can reasonably be expected to involve differences in feeding practices and intakes of dietary iron (Greene-Finestone et al., 1991; Lehmann et al., 1992). 2.9.4 Primary Milk Feeding as a Predictor of Risk for Iron Deficiency Numerous studies have shown a strong association between the risk of iron deficiency and the duration of breast-feeding and the age of introduction and extent of feeding of infant formulas or cows' milk. Numerous clinical studies have shown the efficacy of iron-fortified infant formula in preventing EDA (Moffatt et al., 1994; Daly et al., 1996; Stevens & Nelson, 1995; Irigoyen et al., 1991). Consistent with this, observational studies have shown that infants fed iron-fortified infant formula as their primary milk feeding have a low prevalence of EDA (Innis et al., 1997; Pizarro et al., 1991). In contrast, feeding with low iron formula for more than 4 mths has been associated with a higher prevalence of EDA (Innis et al., 1997). Feeding with iron-fortified, soy protein-based formula has also been shown to be as effective as iron-fortified cows' milk protein-based infant formula in the prevention of EDA during infancy (Hertremph et al., 1986). Early introduction (prior to 6-8 mths of age) (Sadowitz & Oski, 1983; Mills, 1990; 53 Chapter 2. Literature Review Tunnessen & Oski, 1987) and high intakes (>1 litre) (Sadowitz & Oski, 1983; Mills, 1990) of unmodified cows' milk has also been associated with an increased risk of EDA. The reasons for this include the low iron content and bioavailabihty of cows' milk, and the risk of occult bleeding from the gut, especially in infants fed cows' milk in the first 6 mths of life (Fuch et al., 1993a; Zeigler et al., 1990; Fomon et al., 1981). Although the high bioavailability of iron in human milk suggests breast-feeding will protect against iron deficiency, breast-feeding beyond 3-6 mths without the introduction of iron containing foods or supplements is associated with EDA (Siimes et al., 1984; Innis et al., 1997; Calvo et al., 1992; Pizarro et al., 1991; Walter et al., 1993; Willows et al., 2000). Innis et al. (1997) found that 9 mth old infants in Vancouver who were breast-fed for more than 8 mths were at the highest risk for EDA, with a prevalence of 15% compared with a prevalence of only 3% among infants who had been breast-fed <3 mths. The lower prevalence of IDA among infants breast-fed <3 mths was associated with bottle-feeding with iron-fortified formula A further 30% of all 9 mth old infants breast-fed for >8 mths had low iron stores without EDA (Innis et al., 1997). At least 5 other large studies have reported similar rates of EDA among breast-fed infants in the 2n d 6 mths of life (Siimes et al., 1984; Calvo et al., 1992; Pizarro et al., 1991; Walter et al., 1993; Willows et al., 2000). Together these studies suggest that current strategies for supporting adequate iron nutrition while maintaining breast-feeding are less than ideal. Several studies have found that mothers in Canada who breast-feed for longer durations tend to be Caucasian and of higher SES (Beaudry & Aucoin-Larade, 1989; Myers, 1983; Williams et al., 1996). However, an association between breast-feeding and EDA has also been found in 9 mth old Cree infants of lower SES in Northern Quebec (Willows et al., 2000). Despite the apparent increased risk of EDA associated with longer durations of breast-feeding, it is important to note that the majority of infants breast-fed beyond 3-6 mths, i.e. 85% did so without developing EDA (Innis et al., 1997). It is not clear why some infants who are breast-fed beyond 3-6 mths develop EDA while others do not, and whether variables such as the age of introduction, duration and quantity of feeding of infant formulas, cows' milk or complementary foods can predict whether a breast-fed infant is at risk of EDA. 2.9.5 Intake of Complementary Foods as a Predictor of Risk for Iron Deficiency The rationale for promoting cereal for iron nutrition in young infants seems to be based on its suitability as a vehicle for fortification (i.e. low cost, shelf stability), ease of preparation, low renal solute load and potential allergenicity, nutritional content and tradition as a weaning food in many populations (Hendricks & Badruddin, 54 Chapter 2. Literature Review 1992; Walter et al., 1993; Krebs, 2000). A large double-blind trial by Walter et al. (1993) found a prevalence of 15% EDA among 8 mth old infants who were breast-fed to 4 mths of age and fed an unfortified infant cereal compared with 3% for those fed an iron-fortified cereal. Similarly, the prevalence of IDA among infants fed low iron formula and unfortified infant cereal was 15% compared with 6% for those fed the iron-fortified infant cereal. By 15 mths of age the prevalence of EDA among breast-fed infants fed the unfortified cereal was 27% compared with 12% among those fed the iron-fortified cereal and 24% among infants fed low iron formula and unfortified cereal compared with 8% among infants fed low iron formula and iron-fortified infant cereal. Walter et al. (1993) reported that breast-fed infants consistently consumed lower amounts of cereal, with a mean of 20 g at 6 mths and 25 g at about 8 mths, than formula-fed infants who, all except for 20%, maintained an intake of 30 g/day from within 3 weeks of the initiation of cereal at 4 mths of age. A large inter-individual variation in the amount of cereal actually consumed among infants may in part explain why 12% of the breast-fed and 8% of the low iron formula-fed infants studied by Walter et al (1993) had developed EDA (Hgb<105 g/L) by 15 mths of age, despite having been fed iron-fortified infant cereal. The intakes of cereal in the latter study, however, may have been higher than would be expected in free-living infants, since the infants were visited weekly by a nutritionist and cereal consumption was encouraged. Information on the intake of iron-fortified cereal among Canadian infants has not been published since the 1991 publication of guidelines by the CPS Nutrition Committee aimed at the prevention of EDA. In 1981, a mean intake of 18-20 g infant cereal/day was reported by Yeung et al. (1981) for infants 3 to 10 mths of age in Toronto and Montreal who consumed cereal. However, approximately 10% of infants 5 to 8 mths and 25% of 10 mths of age were not fed infant cereals. Similarly, studies in the U.S. found that only 73% of infants 6-12 mths of age were fed infant cereals, with an average consumption of 19 g/day for these infants (Gerber Infant Nutrition Survey, 1989). The large inter-individual variation in the amount of cereal consumed by infants (Yeung et al., 1981; Gerber Infant Nutrition Survey, 1989; Walter et al., 1993), in addition to the poor bioavailability of the iron are reasons to question the efficacy of iron-fortified infant cereal as a public health strategy for the prevention of EDA (Fomon, 1987; Canadian Task Force on Periodic Health Examination, 1979; Fuch et al., 1993b). Studies have reported an increased risk of EDA among infants fed iron-fortified cereals for < 6 mths (Lehmann et al., 1992) and <3 mths (Greene-Finestone et al., 1991) compared with those fed iron-fortified cereals for longer durations. The results of several studies suggest that late introduction (>9 mths) (Requejo et al., 1999) and inadequate intakes of MPF (Mira et al., 1996; Engelmann et al., 1997) may be important predictors of risk iron deficiency. 55 Chapter 2. Literature Review Using a case-control design, Mira et al. (1996) found that the mean daily intake of heme, but not non-heme iron, was lower in iron depleted than in iron replete infants (0.28 mg/day and 0.42 mg/day, respectively). Similarly, Engelmann et al. (1997) demonstrated that despite similar intakes of total iron, infants who consumed 27 g meat/day maintained their Hgb concentrations, whereas infants who consumed 10 g of meat/day had a significant decreases in their Hgb. Regular consumption of non-fortified complementary foods containing meat have also proved to be effective in preventing iron deficiency in infants fed low-iron formula (Haschke et al., 1988). In contrast to these studies, Lwanga (1996) found no association between the ages of introduction of iron-fortified infant cereal or meats and the prevalence of EDA among 9 mth old infants (Lwanga, 1996; Innis et al., 1997), however, no data on the durations of feeding or the quantities of cereals or meats consumed among the 9 mth old infants were collected. Thus, whether inadequate quantities or duration of feeding are the reason that some breast-fed infants develop EDA while others do not is unknown. 2.10 Strategies for Prevention and Detection of Iron Deficiency in Infancy There are essentially 2 approaches for addressing the problem of EDA in infancy: primary and secondary prevention. Primary prevention involves providing additional iron to the population at risk, whereas secondary prevention involves the early detection of infants at risk for EDA through screening and subsequent iron therapy. Considering the uncertainty over whether the effects of EDA on cognitive development are fully reversible, primary prevention of EDA is indisputably the safest and most prudent approach. However, identification of infants at risk for IDA is also important, particularly if those at risk can be identified prior to the onset of anemia. 2.10.1 Strategies for Primary Prevention of IDA Several primary prevention strategies can be used to address IDA in a developed country such as Canada: increasing the iron content of the diet by fortifying selected food products, providing iron supplements to individuals at risk (Yip, 1997), and decreasing behaviors that place individuals at risk The success of these approaches depends on the strategies available for ensuring that the recommended feeding practices and food sources of iron are affordable, accessible and culturally acceptable for all parents, acceptable to all infants, and that the strategies are effective in preventing EDA. Interventions based on food fortification are feasible in settings where the use of 56 Chapter 2. Literature Review commercially prepared products for infant feeding is common Iron supplementation is appropriate where iron-rich or iron-fortified complementary foods for infants are not available or affordable (Yip, 1997). As a public health strategy, routine supplementation of all infants with iron may not be appropriate. Supplementation with 3 mg ferrous sulfate/day in infants with normal iron stores has been found to result in an increased incidence of infection and reduced growth (Idradinata et al., 1994). Further, although not thought to be a concern during infancy, the prevalence of heterozygous idiopathic hemochromatosis is estimated to be one in 300 among individuals of Caucasian ancestry and increasing iron intake has been shown to accelerate progression of the disease (Cook et al., 1992). The risks involved with supplementing iron replete infants could be avoided by supplementing only those identified with a proven deficiency (Cook et al., 1992), however, strategies for identifying infants with poor iron status are currentiy lacking. Although strategies for appropriate fortification of foods with iron are safe and cost effective, current strategies may not be adequately addressing the problem of IDA among certain groups of infants in Canada. Primary prevention efforts aimed at decreasing the prevalence of IDA in Canada have included the fortification of infant cereals and formulas with iron, and the education of parents and medical professionals to promote exclusive breast-feeding for at least 4 mths, use of iron-fortified formula for infants not breast-fed, or in those receiving formula as well as breast milk, introduction of iron-fortified infant cereals at 4-6 mths of age, introduction of cows' milk not prior to 9-12 mths of age, and continued use of iron-fortified foods beyond one year of age (CPS et al., 1998). While the WIC program in the U.S. provides iron-fortified infant formula and cereals directly to infants from disadvantaged family backgrounds, Canada has no universal program that provides iron-fortified products to infants at risk for IDA. Although recent studies suggest that, in general, infants are fed according to current feeding guidelines (Williams et al., 1996; Kwavnick et al., 1999), the high prevalence of IDA among certain subgroups suggests that the CPS guidelines may not be being followed among certain groups of infants. However, recent data on the feeding practices of infants at risk for poor iron status are lacking. This information is needed to improve infant feeding recommendations and public education aimed at the prevention of IDA. 2.10.2 Strategies for Secondary Prevention of IDA The Canadian Task Force for Periodic Health Examination (1994) concluded that there is insufficient evidence to recommend the inclusion or exclusion of routine Hgb determination for infants not considered being 57 Chapter 2. Literature Review "high risk". However, the Task Force recommended that physicians take particular care to determine the nutritional intake of infants at high risk and consider screening with a blood test at 6-12 mths of age, perhaps optimally at 9 mths of age (Canadian Task Force for Periodic Health Examination, 1994). "High-risk" infants include infants of low SES, Chinese or aboriginal ethnic origin, low birth weight (<2500 g), and infants fed only cows' milk during the first year of life. These "high risk" groups were defined on the basis of a higher prevalence of EDA and a greater likelihood of inability to consume iron-fortified products (Canadian Task Force for Periodic Health Examination, 1994). Similarly, CPS et al. (1998) recommend that a blood test be done for infants 6 to 8 mths of age for whom parents choose not to adhere to the current feeding guidelines. Currently, no standardized, validated method of assessing an infant's risk of IDA based on feeding is available to determine the need for a blood test as recommended by CPS et al. (1998) and the Canadian Task Force for Periodic Health Examination (1994). Strategies for the early detection of infants at risk for EDA could play an important role in reducing EDA by enabling identification of infants prior to development of anemia and allowing resources to be targeted to the infants most at risk. There are potential problems, however, with the approach of early detection of infants at risk for EDA. Clearly, accurate identification of infants at risk of EDA, appropriate investigative follow-up, the effectiveness of subsequent prevention or therapy are essential (Beaglehole et al., 1993; Sackett, 1975). The method of identification should result in few false positives, and more importantly, few false negatives. Although assessment of the infant diet may provide a means of predicting risk for EDA (Pizarro et al., 1991; Boutry & Needlman, 1996), routine screening of the diet as a first stage, followed by a blood test for high risk infants may not be the usual practice of all physicians. Most screening programs are carried out sporadically and rarely capture more than 5-10% of those eligible (Sackett, 1994; James et al., 1997). Mills (1990) and James et al. (1997) provided evidence that the invasiveness of a blood test was not acceptable to all parents with an infant who otherwise appeared healthy. Moy & Auckett (1997), however, have suggested that a screening program aimed at 21 mth old children deemed by socio-demographic and ethnic minority factors to be at high risk for EDA was highly acceptable to 64% of the parents of the infants the program reached. Thus, a blood test following and based on assessment of the adequacy of diet may be acceptable for Canadian parents and physicians. Currently in Canada, infants with EDA are identified in a clinical setting if EDA is suspected, or from the results of a blood test for problems unrelated to the EDA. The usual practice is to screen with a CBC and, in the case of abnormal red cell indices indicative of anemia, confirm the diagnosis by measurement of ferritin or, alternatively, a trial of iron therapy (Canadian Task Force on Periodic Health 58 Chapter 2. Literature Review Examination, 1994). For public health purposes, a dietary assessment tool to detect infants at high risk for EDA should be easy to administer, simple for parents to fill out, or health professionals to administer, reproducible and accurate. Boutry & Needlman (1996) assessed the usefulness of a brief dietary history as a screening tool for microcytic anemia (Hgb <110 g/L and M C V <75 fL) in a group of low-income, African-American infants aged 13 to 60 mths who had previously received nutritional support from the WIC Program In the latter study, infants were classified retrospectively, based on documentation of a brief dietary history taken in the course of primary care visits, as 'dietary deficient' if they ate <5 servings each of meat, grains, vegetables and fruits/week, drank >16 ounces of milk/day, ate any fatty snacks or sweets, or drank >2 glasses of pop/day. In this study, 8% were found to have microcytic anemia This classification of dietary deficiency had a sensitivity of 71% and a specificity of 79%, although the negative predictive value was 98%, and the positive predictive value was only 9%. The dietary screening tool of Boutry & Needlman (1996) was recently re-evaluated in a parent-completed dietary and health history format for infants 9-30 mths in inner city clinics in Baltimore City. This study by Bogen et al. (2000) also evaluated 15 other dietary items in the domains of infant diet, intake of solid food, intake of beverages, and participation in the WIC Program, together with 14 historical items in the domains of birth history, past health, and maternal and family history. Neither individual nor combinations of parental answers were able to predict EDA, anemia, or iron depletion well enough to serve as a screening test. A nutritional screening tool called the PEACH survey developed to detect children with nutrition or feeding problems from birth to 5 years was designed to detect behavioral feeding problems, rather than iron deficiency (Campbell & Kelsey, 1994). Whether a brief dietary assessment tool, and the dietary factors that should be included could be of value for detecting infants from Caucasian and Chinese ancestries at risk for EDA, is not known. Development of such a tool first requires a better understanding of the current dietary factors and feeding practices associated with EDA among Caucasian and Chinese infants at risk for EDA. 2.10 Research Instruments for Assessing Dietary Intakes in Infancy Published information on the development and use of dietary assessment instruments in infancy and early childhood is limited, particularly in the area of iron nutrition Although there is an abundance of comparative 59 Chapter 2. Literature Review studies, there are no universal criteria for selecting the most appropriate dietary assessment instrument for use in infancy (Frank, 1994; Willett, 1998). In general dietary assessment instruments fall into 2 categories: those based on memory (e.g. 24-hour recall, food frequencies and dietary histories) and those based on recording of actual food consumed (e.g. direct observation and dietary records). Methods based on the direct observation or recording of food consumed are better suited to providing quantitative information on an individual's intake and are considered the gold standard against which other methods are compared (Thompson & Byers, 1994; Willett, 1998). The selection of dietary assessment instruments requires consideration of factors such as the need for surrogate reporting, participant access and burden, and the usual practices and daily routines of the participants. Rapid changes in feeding practices and food habits and a high degree of intra-individual variability that increases with age (Black et al., 1983; Miller et al., 1991; Harbottle & Duggan, 1994) are characteristic of infancy, and pose additional challenges for measuring dietary intakes in this age group. Unless direct observation is being used for dietary assessment, data from other sources including surrogate reporters (often more than one and both within and away from home) is necessary (Rockett & Colditz, 1997; Baranowski, 1994; Frank, 1994) for infants. Determining the intake of breast milk in the breast-fed infant poses additional challenges. Estimating the intake of table foods may not be straightforward either as infants may eat only a small portion of the food offered. Establishing the validity and reliability of dietary intakes is often a difficult task since a "true picture'' of a child's intake is often beyond reach (Persson & Carlgren, 1984). Direct observation of dietary intakes involves considerable cost and effort and may introduce a social-desirability bias. The dietary record methodology is well-known to underestimate intakes (Thompson & Byers, 1994), requires a large number of days to quantify intakes, and is associated with increased cost, time, poor response rates, and decreased quality of recording as the number of days increase, particularly towards the end of a recording period (Persson & Carlgren, 1984). Dietary records may also cause changes in the types and quantity of food eaten (Barrett-Connor, 1991). In addition, dietary records are subject to bias towards more motivated and literate participants (Harbottle & Duggan, 1994). Dietary records, particularly those that are weighed and/or for greater than 3 days duration, have the highest refusal rate and highest percentage of subjects with unusable data (Willett, 1998). Generally, one or more days with a sample of at least 60 subjects, however, can adequately characterize a group (Farris & Nicklas, 1993). Training by both participant and interviewer, and contact and review of the dietary record on the 2n d day of recording and one day following recording to clarify and probe for forgotten foods has been shown 60 Chapter 2. Literature Review to enhance the accuracy of food records (Bolland et al., 1988). Because of this, dietary records are very labor intensive in terms of adnumstration, quality control and data entry, and as a result, are very costly. Despite these limitations, dietary records are typically considered to be the referent standard for data on the intake of many nutrients (Jacques et al., 1993). Diet histories can be used to determine usual food intakes, providing details about the characteristics of foods as consumed, and the frequency and amount of food intake. However, diet histories traditionally are meal-based, and this has limited use in infancy when defined meals are not usually eaten. The brief dietary assessment is another method that can be used to determine specific food consumption behaviors. Such measures are not quantitatively meaningful, do not encompass the whole diet, and do not allow estimates of dietary intake. Despite this, brief assessment methods have considerable application for deterrnining feeding practices and food consumption patterns in infancy. Food frequency questionnaires (FFQs) and 24-hr recalls have the disadvantage of the potential for individuals to inaccurately report food consumption for reasons related to memory and the interview situation An assessment of the agreement between 12-hr observation and 12-hr recall reports of dietary intake and the mother's monthly reports of usual feeding practices in 131 low-income Peruvian infants found that consumption during the past 12-hr observation and 12-hr recall was not an accurate method to classify infant feeding practices. Exclusive breast-feeding in infants younger than 4 mths was observed 25% more often than reported, while non-human milk consumption was reported 30% more often than observed. Most of the disagreement between reported and observed practices could have been due to daily variations in feeding practices as the time periods of measurement differed (Piwoz et al., 1995). The authors suggested that single day studies may be misleading and that the current WHO recommendations regarding the use of single day, 24-h recall methods to assess infant feeding practices be reconsidered. The use of food frequency or diet history questionnaires may have a higher degree of reliability and validity, be easier to aclminister and more accurate (Willett, 1998). FFQs minimize respondent and adrninistrative burden, are useful for describing average dietary intakes and ranking individuals according to their usual consumption of foods, groups of foods, and nutrient intakes, and for assessing the association between dietary intake and disease (Willett, 1998; Frank et al., 1992; Rockett et al., 1995; Blom et al., 1989). However, FFQs do not provide an accurate estimate of actual levels of intakes (Thompson & Byers, 1994). Comparison with dietary records is considered to be the best way to establish the relative validity of a FFQ because these 2 dietary assessment 61 Chapter 2. Literature Review methods are likely to have the fewest correlated errors (Willett et al., 1985). However, the tendency to misreport food intake and the errors inherent in the use of food composition data may be similar for both FFQs and 3d-FRs. The potential for correlated errors associated with the estimation of iron status from dietary and biochemical measures are much more likely to be independent (Jacques et al., 1993; Willett, 1998), although there are also a number of limitations associated with biochemical validation of dietary instruments. Dietary and biochemical indices measure 2 different things, the intake and circulating concentrations of a nutrient, respectively. Since biochemical indices of iron status are influenced not only by iron intake but other dietary and non-dietary factors, a correlation between a reported iron intake and an objective biochemical marker of iron status can be interpreted as the lower bound of the true questionnaire validity (Jacques et al., 1993). Rapid growth and development are characteristic of infancy and early childhood and, result in greater intra-individual variations in food and nutrient intakes than for adults (Miller et al., 1991). Intra-individual variability in intakes influences the number of days required to accurately estimate food and nutrient intakes. In early infancy, when breast milk and infant formulas are the primary milk feedings, the variability in nutrient intakes is low. Variability can be expected to increase, however, with the progression of weaning and the introduction of complementary foods (Black et al., 1983). Based on weighed intake data from a one year cross-sectional survey of Indo-Asian children (4-40 mths of age), it has been estimated that 3-5 days of recording are needed to classify children for iron intakes, while 2 days are needed to classify children for vitamin C intakes (Harbottle & Duggan, 1994). Research concerning iron nutrition in infancy and detection of infants at risk for iron deficiency has been hampered by the lack of dietary assessment instruments that measure the dietary intakes and sources of iron and inhibitors and enhancers of iron absorption, and are predictive of an infant's risk of IDA. Although FFQs for assessing iron intake have been developed and validated for use with adults (Willett et al., 1987), studies on the use of FFQs during infancy have not been published. 62 Chapter 3. Design and Methods C H A P T E R 3. DESIGN A N D M E T H O D S 3.1 Study Design and Ethical Approval This was a cross-sectional study of the feeding history, food and nutrient intakes and socio-cultural background of full-term infants 8-26 mths of age from Caucasian and Chinese ancestries in relation to their iron status. The study protocol and procedures were approved by the University of British Columbia (U.B.C.) Screening Committee for Research involving Human Subjects (Appendix A). 3.2. Development and Use of Dietary Assessment Instruments For the purpose of this study, 3 dietary assessment instruments were developed. These instruments were a 36-item, 13-page Socio-Cultural and Infant Feeding Questionnaire (Appendix B), a 3-day Food Record (3d-FR) Package (Appendix C), and a 191-item, 25-page Food Frequency Questionnaire (FFQ) for Parents of Infants 8 to 26 mths of age (Appendix D). The Socio-Cultural and Infant Feeding Questionnaire was developed to examine the relations between feeding history and socio-cultural background with the risk for EDA and low iron stores. No FFQs designed to assess iron nutrition are currently available; thus the FFQ was developed to fill this gap in pediatric dietary assessment methodology. The FFQ was designed to identify trends in major food group consumption and to examine the value of the assessment of the intakes and food sources of energy, iron and other dietary factors influencing iron absorption to identify infants at risk for IDA. The 3d-FR was chosen as the best available comparison dietary assessment method, and for the purpose of quantifying dietary intakes of iron (total, heme and non-heme) and other dietary factors associated with risk of EDA and low iron stores. These dietary assessment instruments were also chosen to achieve a balance between minimizing respondent burden and maximizing the validity and precision of the assessment of food and nutrient intakes. 3.2.1. Food Frequency Questionnaire (FFQ) An mterview-adrninistered FFQ was developed for the purpose of collecting information on the dietary intakes and consumption of foods from food categories representing the major sources of iron and other dietary factors that influence iron absorption, as well as provide a comprehensive assessment of energy intake. The FFQ was developed to be culturally relevant for infants 8 to 26 mths of age from Caucasian and Chinese ethnicities, and from vegetarian and 63 Chapter 3. Design and Methods non-vegetarian families using techniques described by Teufel (1997) for improving the cultural competency of a dietary assessment tool. Input from the target groups was obtained during the development of the FFQ for the purpose of compiling a culturally relevant food list, identifying culturally specific food preparation techniques and recipes and culturally appropriate portion sizes, and to enable development of a odturally-specific food composition database. Face validity was addressed by consultation with Dietitians/Nutritionists from the Vancouver Health Department and British Columbia's Children's Hospital who work with Caucasian, Chinese and vegetarian families. Then, the FFQ was pre-tested with 12 parents who represented the target groups. The parents who participated were asked to complete the FFQ, then give their input on the appropriateness of food items, food groups and portion sizes. Following this, a pilot study (below) was conducted, during which the FFQ and a 3d-FR were completed by 30 parents who again represented the target groups of the study. Research nutritionists who could speak Cantonese, Mandarin and English were employed to increase the cultural competency of the data collection and analysis. The FFQ consisted of 191-items considered to represent the major dietary sources of energy, iron and dietary factors that influence iron absorption and that would be eaten by infants of 8-26 mths of age. The following approaches were used to select food items for inclusion in the FFQ: 1) foods high in iron and containing factors known to influence iron absorption were taken from food lists from existing Canadian (i.e. Ontario Health Survey FFQ, Bright-See et al., 1994) and U.S. (i.e. Harvard University FFQ, Harvard Eating Survey for Children, National Health and Nutrition Examination Survey EI FFQ, Thompson & Byers, 1994) FFQs, published literature (Monsen & Balintfy, 1982; Monsen et al., 1978; Fairweather-Tait, 1989; Hazell, 1985; Lynch & HurrelL 1990) and food composition tables (Pennington, 1994; Holland et al., 1990; Stewart & Stewart, 1990; Health & Welfare Canada, 1989), and 2) foods and food categories that are lower in iron and factors known to influence iron absorption but contribute to total energy and nutrient intakes, irrespective of their relevance to iron nutrition Examples of foods and/or brand names were provided with each food item in the FFQ to help clarify for the parents what foods should be included in that category. An open-ended question at the end of each food group section allowed parents to report any other foods eaten but not listed on the questionnaire. The FFQ was designed to record if a particular food had been consumed in the preceding 2 weeks, and if so, the number of times per day or per week, and the quantity usually eaten. Portions were recorded as one of 3 appropriate standard size servings, e.g. whole milk: 'A cup (2oz), ¥z cup (4oz), and one cup (8oz). An open box was also provided to allow recording of a portion size other than those given The FFQ also included questions to obtain the following information: 1) whether or not the infant was breast-fed or fed a human milk substitute at the time of the study, 2) the 64 Chapter 3. Design and Methods frequency and usual duration of any breast-feedings, 3) the frequency and amounts of any human milk substitutes fed, 4) the types of milk feedings fed during each mth from birth to the time of the study, 5) the use of vitamin and mineral supplements given since birth to the time of the study. A Microsoft Access (Version 2.0, Redmond, WA) database was specifically designed to analyze the dietary information collected using the FFQ through consultation with the Centre for Evaluation Sciences at B.C.'s Children's Hospital and ESHA Research (Salem, Oregon). This database is referred to as the "FFQ Analysis Database". The FFQ Analysis Database was designed to enable entry of the FFQ data as recorded, thus preserving data on usual food consumption frequency and portion sizes, and to create a database that could be imported into the Food Processor (FP®) for Windows Nutrition Analysis & Fitness Software (ESHA Research, Salem, Oregon) database for calculation of average daily food and nutrient intakes. 3.2.2. 3-day Food Record (3d-FR) Package A 3d-FR was developed to collect detailed information on each infant's dietary intake during a 3-day period This package had 4 components: 1) Guidelines on Keeping a Food Record, 2) Food Record Examples, 3) 3 Blank Food Record Forms, and 4) Portion Size Tools (Appendix C). The guidelines included instructions on how to record the times and places of eating, and how to describe the foods and beverages eaten, and the quantities (volume, weight or size) actually eaten Parents were also asked to record the type(s), brand name(s) and the amounts) of any vitamin and/or mineral supplements given, to record if this was a typical day for their child and if not, the reason(s), and to provide any other relevant comments. 3.2.3. Food Composition Database The FP® was chosen as the nutrient analysis program because it contains an extensive database of Canadian and infant foods and has been rated as one of the best for research purposes (LaComb et al., 1992; Lee et al., 1995). The food composition data used in this study were derived primarily from the Canadian Nutrient File Release (1997) with some additions from USDA NDA Release No. 11, and is abbreviated as the ESHA Database. There were approximately 250 foods recorded on the 3d-FRs for which there were no comparative foods in the ESHA Database. Food composition data for these foods were obtained from Pennington (1994), Chinese Food Composition Tables (Stewart & Stewart, 1990) or the manufacturers. The food was then assigned a USER code, and the nutrient composition was added to the 65 Chapter 3. Design and Methods FP® database. Another 146 composite food items recorded on the FFQ could not be adequately represented by single, pre-existing foods in the ESHA database. Thus, a list of foods that represented these food items was created and was termed "FFQ composite'' food list An example of a FFQ composite food list for the food item "Mixed Dishes made with Beef is shown in Appendix £ . The analysis of each FFQ composite food provided nutrient composition data, and this nutrient composition was then assigned a USER code and added to the FP® database (Appendix E). A complete list, including the assigned USER codes of all foods for which food composition data were generated and then added to the database is shown in Appendix F. 3.2.4. Socio-Cultural and Infant Feeding Questionnaire The Socio-Cultural and Infant Feeding Questionnaire was developed for the purpose of collecting information on the family socio-cultural background and the infant's nutritional history. This questionnaire was based on 2 questionnaires that had been designed to collect information on family background and nutritional history in previous research with infants at 9 mths of age (Lwanga, 1996). A socio-cultural section of the questionnaire was designed to collect information on demographic characteristics and family dietary practices that might allow for identification of infants at risk for IDA or low iron stores. This section included questions on the parent's age, present living arrangement and marital status, highest level of education attained, usual occupation, ethnic background, race, number of children in the household, annual family income, the number of years the parents had lived in Canada, language spoken at home, ethnic food practices, foods excluded from the family diet, and any special dietary practices. The infant feeding history section of the questionnaire was designed to collect retrospective and current information that would allow for identification of feeding practices that might be associated with IDA or low iron stores. This section included questions on the duration of breast-feeding, the age of introduction and types of infant formula (low iron or iron-fortified), cows' milk (whole, 2%, 1%, or skim) or other milks (goats' milk, soy milk, etc.) fed from birth to the time of the study, and the types and amounts of infant formula(s), cows' milk or other milk(s) currently fed. The infant feeding section also included questions on the age of introduction, duration of feeding and, where applicable, reasons for stopping feeding of complementary foods including cereals, rice, pasta, breads/crackers, vegetables, fruits, legumes, dairy, other animal products (e.g. eggs, meat, poultry or fish) and fruit juices. This questionnaire also had questions on the type(s) of infant cereal(s) introduced, the hquid(s) used to prepare infant cereals, and for those who had not yet introduced infant cereal, the reasons for not introducing an infant cereal. Parents were also asked to rate the nutritional quality of their infant's 66 Chapter 3. Design and Methods diets, and eating habits, and provide information on childcare outside the home and the individuals involved in the infant's food preparation A Microsoft Access (Version 2.0, Redmond, WA) Database was designed in consultation with the Centre for Evaluation Sciences at B.C.'s Children's Hospital specifically for analysis of the Socio-Cultural and Infant Feeding Questionnaire that would enable entry of the data as recorded and allow transformation to a computer spreadsheet 3.2.5. Validating the Dietary Assessment Instruments Several steps were taken to increase the face and content validity of the instruments. Local experts in nutrition and pediatrics, i.e., Vancouver Health Department Public Health Nutritionists, B.C.'s Children's Hospital Department of Nutrition Services, and Dr. Susan Barr, Faculty of Agricultural Sciences, U.B.C. were consulted to review the content validity of the questionnaires. Experts in survey design and statistics, Ruth Milner, Health Care & Epidemiology, U.B.C., and Thomas Lam (Computer Programer) and Laurie Ainsworth (Statistician), Centre for Evaluation Sciences, B.C. Research Institute for Children's & Women's Health were consulted to review the design and analysis of the dietary assessment instruments. These reviews were used to further refine the dietary assessment instruments. All the instruments were translated into Chinese (Appendix G , H , I), and then back into English by an independent, 2n d individual to ensure accuracy of the translation and cultural-specificity. The instruments were then pre-tested with 12 parents representative of the target populations to address face and content vakdity, and to ensure that they were clear. Parents were asked to complete the questionnaires and to give constructive criticism on content, clarity and appropriateness of the questions, their willingness to complete the questionnaires, and if there was anything they thought should be added This pre-test led to several changes in the questions and wording. The revised questionnaires were then circulated for comments to the City of Vancouver Public Health Nutritionists and final revisions were made. The research nutritionists employed to assist with this study were trained to ensure consistent adininistration of the research instruments. Three of the research nutritionists involved in the data collection, development of the culture-specific food composition database and the data entry phases of this study were of Chinese ancestry and were fluent in Cantonese and Mandarin, in addition to English. A pilot study with a sample of 30 parents with infants of similar age and with a similar socio-cultural background to those who would be included in the study population was undertaken from August 1995 to January 1996. The purpose of the pilot study was to ensure that the dietary assessment instruments and study protocol could 67 Chapter 3. Design and Methods be understood and completed by the target groups. The intent of the pilot study was to identify areas of difficulty and ensure that the experience of completing each phase of the study was a positive one. Parents for participation in the pilot study were recruited from parent and infant/toddler groups offered at health units, community centres and neighbourhood houses throughout the City of Vancouver. Recruitment sites for the pilot study included Sheway Community Project for Women and Children, UBC Family Housing, and the North and South Health Units of the Vancouver Health Department. A 24-hour recall was conducted by interview with the parent and recorded on a blank food record form This 24-hour recall procedure was used to instruct the parents on how to keep the 3d-FR During the following week, the parents completed a 3d-FR. One week later, the FFQ was completed by a face-to lace interview. Using this protocol, the 2 weeks covered by the FFQ included the 3 days over which the 3d-FR was recorded. The reliability of the dietary assessment instruments was not formally assessed. 3.3 Subjects 3.3.1. Participant Identification and Selection Criteria A sample of infants for participation in the study was systematically identified using birth lists provided by the City of Vancouver Public Health Department Parents' names, addresses, phone numbers and the infant's name and date of birth were provided on the birth lists. Infants who would be 9±1, 15±2 or 24±2 mths of age during the dates of the scheduled research clinics were identified from the birth lists. Infants were recruited from these 3 age groups to examine the risk of IDA and low iron stores by age from about 8 to 26 mths of age. Infants from Caucasian and Chinese family backgrounds were targeted using the family surname given on the birth list. The selection criteria were that the infant was bom to parents resident in Vancouver, with an address to enable contact Exclusion criteria included prematurity (gestational age <37 weeks), birth weight <2500 or >4500 g, history of serious or confounding illness (e.g. sickle cell anemia, blood clotting disorders, chronic bowel disease, liver disease, endocrine deficiency, blood loss, hemolytic disease, bone marrow depression, polycythemia, erythrocytosis, exposure to lead or other toxins, malignancy, chronic or congenital disorders), or a history of major surgery. For the purpose of this study, birth weights were rounded to the nearest 50 grams. Infants were also excluded if the parent was unable to speak sufficient English, Cantonese or Mandarin to allow competent completion of the informed consent and study instruments. 68 Chapter 3. Design and Methods 3.3.2. Participant Recruitment A total of 1585 infants who would be 8-10, 13-17 or 22-26 mths of age during the dates of the scheduled research clinics were identified from the birth lists and assigned a 4-digit identification number. A letter in English and Chinese (Appendix J and K) was mailed to the parents of all these 1585, potentially eligible infants, using the address on the birth lists. This method of recruitment was chosen because telephone is not acceptable to the Vancouver Health Department or the U.B.C's Screening Committee for Research Involving Human Subjects as a method for first contact of research study participants. The letter sent described the study and invited parents to participate by attending a clinic offering assessment of their infant's diet and iron status. The letters were sent about 2 to 3 weeks before a potential nutrition research clinic appointment The letter was followed by a telephone call by a trained research nutritionist approximately one week later to ask if the parent had received the letter and if they were interested in participating in the study. If the parent was interested, the ehgibility criteria were reviewed with the parents. If the infant met the eligibility criteria, the requirements for participating in the study were described in detail. If the parent agreed to participate, an appointment was made to attend a scheduled clinic at a time suitable for the parent. At least 3 attempts were made to reach each infant's parents. No attempt was made to contact parents for whom a letter had been returned due to a wrong address because U.B.C. ethical approval required that parents be contacted by letter first Eligible infants were also recruited from parent and infant/toddler groups held at health units, community centres, neighbourhood houses and immunization clinics in the City of Vancouver through collaboration with the Vancouver Health Department Public Health Nutritionists and Nurses. This allowed participation of infants meeting the eligibility criteria for whom the parents had not received the study letter. The parents attending the infant/toddler groups were asked by the Public Health Nutritionist or Nurse running the group if they would be interested in receiving information on an infant nutrition study. Upon the group's approval, the study was explained in person to the parents by the research nutritionist (PLW), and parents with eligible infants were invited to participate. Parents who had made an appointment to attend a nutrition research clinic were telephoned one-day prior to the scheduled appointment as a reminder. At that time, the parent was asked if the infant had had any illness within the previous week, was taking any medications (e.g. antibiotics), or if there was any reason they could not attend the appointment Those infants who had been ill in the previous week, were taking medication, or were unable to attend the appointment were rescheduled where possible. The infant's physician's name, telephone number and mailing address, birth anthropometric measures and parent's mailing address were requested and recorded on the personal data form 69 Chapter 3. Design and Methods (Appendix L) during the telephone call just prior to the scheduled appointment If the parent was not able to provide the information during the telephone call they were asked to bring it to the clinic appointment 3.3.3. Clinic Scheduling Research clinics were set up at health units, community centres and neighbourhood houses at various locations throughout Vancouver to facilitate recruitment of a representative sample of the study target populations. Initially a series of 6 clinics was scheduled from February to March 1996. These clinics were at West Main Health Unit (February 6*, 19%), Kitsilano Community Centre (February 24th, 19%), Brittania Community Centre (March 9th, 19%), East Health Unit (March 18th, 19%), South Health Unit (March 22nd, 19%), and Mount Pleasant Health Unit (March 27th, 19%). A 2n d series of 5 clinics was scheduled from May to July 19% to facilitate a greater representation of Chinese and vegetarian families. This 2nd series of clinics was held at South Health Unit (May 31st, 19%), Kiwassa Neighbourhood House (June 8*, 1996), East Health Unit (July 10th, 1996), South Health Unit (July 12*, 19%) and Kitsilano Community Centre (July 20* 19%). Parents willing to participate but unable to attend a scheduled clinic were seen either in their home or at B.C.'s Children's Hospital. Clinics were scheduled as a full-day or half-day (morning, afternoon or evening), on week days, or as a morning or full-day clinic on weekends, each lasting 3-11 hours, with one infant seen each half-hour. Refreshments and volunteer childcare were provided Transportation to and from the clinics was offered for parents without available transportation. 3.4. Data Collection 3.4.1. Socio-Cultural and Dietary Data At the clinics, the study was explained to parents and informed, written consent was obtained (Appendix M). The 4-digit number previously assigned to each infant in the recruitment process was used on all blood tubes and the Socio-Cultural and Infant Feeding Questionnaire to ensure confidentiality of the laboratory and socio-cultural data. Any personal data not previously obtained was collected and recorded on the personal data form (Appendix L). Parents were then asked to complete the Socio-Cultural and Infant Feeding Questionnaire. The socio-cultural section of this questionnaire was not reviewed with the parents after completion for reasons of confidentiality. Following this, a trained nutritionist conducted a 24-hour dietary recall of the infant's intake during 70 Chapter 3. Design and Methods the previous day (12:00 am to 11:59 pm) with the parent by a face-to-face interview and recorded the information on a blank food record form. This 24-hour food recall procedure was used to instruct the parents on how to keep the 3d-FR through illustration of how to record all foods and beverages consumed and how to describe items and portion sizes in sufficient detail. Food pictures, plastic food models, measuring utensils and containers of common commercial infant foods and serving dishes were used to instruct the parents on how to record portion sizes. Parents were then instructed to keep a record of all foods and beverages consumed by their participating infant over 3 consecutive days, one of which was to be a Saturday or Sunday. Parents were asked to provide detailed descriptions, including brand names, methods of preparation and recipes whenever possible, of all foods and beverages consumed by their infant. Parents were given the 3d-FR package, standardized plastic measuring utensils and weighing scales to take home and asked to record their infant's intake on the 3d-FR forms during the week following the first research clinic appointment. One week later, the parents attended a 2n d follow-up nutrition research clinic, or they were seen at the parent's home or the B.C.'s Children's Hospital. At this time, the 3d-FR was reviewed in detail with the parent for clarity and missing information. Then, the FFQ was administered to the parents by a trained nutritionist in a face-to-face interview, usually taking 30-70 minutes. 3.4.2 Anthropometric Measures Anthropometric measures were obtained for all participating infants by one of 3 trained personnel at the first clinic appointment, with the parent present at all times. The measurements made were body weight, length and head circumference. These measurements were obtained to allow consideration of potential confounding effects of delayed growth in any infants found to have IDA. Infants with a body weight for length below the 5th percentile, based on the National Centre for Health Statistics percentiles (Hamill et al., 1979) were considered at potential risk for delayed growth. All of the anthropometric measures were recorded directly on the personal data form when they were taken 3.4.2.1. Body Weight The parent was asked to undress the infant but leave a dry diaper on (dry diapers were available if needed). The weight of the infant was then measured in duplicate. A 3rd measurement was taken if the first 2 measures differed by more than 10 g. Body weight was measured using an electronic balance accurate to 5 g (Digital baby scale model 727, Lux & Zwingenberger LTD, Lakeshore, Toronto) and immediately recorded, without adjusting for the weight of the diaper. 71 Chapter 3. Design and Methods 3.4.2.2 Length Crown to heel length was measured in the recumbent position using a pediatric length board (Ellard Instrumentation LTD, Seattle, WA) to the nearest mm in duplicate. A 3rd measurement was made if the measures differed by more than 2 mm 3.4.2.3 Head Circumference Head circumference was measured using a disposable paper tape (Mead Johnson, Evansville, IN) placed over the part of the occiput which gives the maximum circumference (Gibson, 1990) to the nearest mm. Two measurements were taken, and if these differed by more than 2 mm, a 3rd measurement was made. 3.4 J Blood Collection A blood sample was obtained from each infant at the first clinic appointment following completion of all of the other measures. The parent was asked to hold their infant on their lap throughout and following the blood collection A trained phlebotomist from B.C.'s Children's Hospital collected 2 tubes of capillary blood from a finger prick made using a sterilized lancet and after warming the infant's finger. First 250 uL of blood was collected into an ethylene diamine tetra-acetic acid (EDTA)-coated microtainer tube for later complete blood count (CBC) analysis. Then, 800 uL of blood was collected into a 2n d microtainer tube coated with lithium heparin for later analysis of serum ferritin and sTfR. The tubes were labeled with the infant's 4-digit study ED number, the date and the clinic number. The EDTA-coated tubes were kept in ice until delivery to the B.C.'s Children's Hospital Hematopathology Laboratory immediately following the clinic. The hthium heparin-coated tubes were kept at room temperature, and taken to the nutrition laboratory at the B.C. Research Institute for Children's and Women's Health following completion of each clime. 3.5 Data Analysis 3.5.1 Pilot Study The foods recorded on the 24-hour recall and 3d-FR were reviewed following completion to determine if the FFQ included all the major sources of energy, iron and dietary factors known to influence iron absorption (meat, fish and poultry, vitamin C, calcium, phytate and fibre). Each 24-hour recall, 3d-FR and FFQ was then analysed using the FP® 72 Chapter 3. Design and Methods (Version 6.03, ESHA Research, Salem, Oregon) to deterrnine the mean dairy intakes of iron and other nutrients of concern (energy, vitamin C, calcium and fibre). The nutrient intakes estimated from the FFQ were then compared with the intakes estimated from the 3d-FR to determine if any important food sources had been omitted from the FFQ. Any additional foods contributing to the intakes of energy, iron or dietary factors known to influence iron absorption (meat, fish and poultry, vitamin C, calcium, phytate and fibre) identified on the 24-hour recall or on the 3d-FR were added to the FFQ. 3.5.2 Socio-Cultural and Infant Feeding Questionnaire The completed Socio-Cultural and Infant Feeding Questionnaires were entered into the Microsoft Access database, then the data were checked by one person to ensure accuracy and consistency in the way decisions regarding the data entry were made. 3.5.3 3-day Food Record and Food Frequency Questionnaire .. The 3d-FRs were coded, entered into FP® and analysed, originally using FP® (Version 6.13, ESHA Research, Salem, Oregon), and subsequently using FP® (Version 7.03). Mixed dishes were disaggregated into their respective ingredients according to recipes provided, and the ingredients entered separately. Mixed dishes for which no recipe was provided were entered as the food from the ESHA database that was most similar based on judgement. After the 3d-FR data had been entered, the food list representing the foods recorded on the 3d-FR for each infant was compared to the original 3d-FR for accuracy by a 2nd person Changes were made to the data entered as needed, and checked again All the completed food lists were checked a final time by the same person to ensure accuracy and consistency of the decisions made in data entry. Some food items had not been described in adequate detail in the 3d-FR provided by the parents. For these foods, the closest possible food was chosen based on the best available data. The FFQs were reviewed, coded and entered into the Microsoft Access (Version 2.0, Redmond, WA) database. Prior to data entry, each FFQ was reviewed for the purpose of categorizing and tabulating all the foods recorded in the 'other foods' options of each major food group section A maximum of 5 of the most frequently reported 'other foods' from each of the 14 food group sections in the FFQ were added as data entry options to the FFQ Analysis Database. All the remaining 'other foods' on each FFQ were categorized into pre-existing FFQ food categories. All of the 191 food items in the FFQ and each of the 72 new foods (n=263) were then assigned either an ESHA code, (i.e. number given in 73 Chapter 3. Design and Methods the FP® database) or a USER code (i.e. a number assigned to represent a food for which the nutrient composition had been added to the FP® database). Of the 191 food items, ESHA codes were used for 117 for which a single food in the ESHA database adequately represented the food, e.g. whole milk. One hundred and forty six foods on the FFQ were not adequately represented by a pre-existing food in the ESHA database. Thus, as previously described, a "FFQ composite", represented an average nutrient composition for foods and mixed meals not in the database, was created and the nutrient analysis for each was added to the FP® database with an assigned USER code. The nutrient compositions of infant formulas not in the FP® database were obtained from the manufacturers and added to the FP® database. The FFQ coded for all food categories is shown in Appendix N. Following entry of all the 3d-FRs and FFQs, FP® Version 7.03 (ESHA Research, Salem, Oregon) that includes the recently released update of the Canadian Nutrient File (1997) and the USDA NDB (Release No. 11) was made available. These more recent food composition databases were clearly more likely to reflect the nutrient composition of the food supply during the study. Therefore, the 3d-FRs and FFQs were imported from Version 6.03 to Version 7.03 of the FP® and each new 3d-FR and FFQ food list generated by FP® was compared to the corresponding original data, and any USER codes incorrecdy assigned were corrected After entry of the FFQ data, average daily consumption data for each infant was calculated to produce a Microsoft® Excel 97 (Microsoft Corporation, USA) database. The Excel database contained the subject numbers, ESHA and USER food codes, portion size codes and average daily amounts consumed for each food, for each infant for the 2 week period recorded on the FFQ. This Excel database was reviewed to ensure that the food items accurately represented the food items in the FFQ. Any coding errors or missing foods were corrected and the Excel database then reviewed a final time. The final Excel database was then imported into FP® using the "ESHAPortO" software (Version 1.0, 1997, ESHA Research). A FFQ food list was generated for each infant from FP®, reviewed and any food items lost during the process of importing the Microsoft® Excel 97 (Microsoft Corporation, USA) FFQ Database into FP® were added. After completion of data entry, each FFQ and 3d-FR was analysed using FP® to calculate the average daily intakes of energy, iron (total, heme and non-heme), vitamin C, calcium and dietary fibre for each infant The intakes of heme and non-heme iron were then estimated To accomplish this, all foods on the FFQs and 3d-FRs were coded as either meat, poultry, fish (MPF), mixed dishes containing MPF, or foods not containing MPF. Information on MPF content of purchased infant foods was obtained from the manufacturers. For other mixed dishes, the MPF 74 Chapter 3. Design and Methods content was estimated based on macronutrient composition data from FP®, then the iron content multiplied by the percent of total iron estimated to be from MPF, to give the estimated amount of iron from MPF. The amount of heme iron was then estimated based on the assumption that the iron in MPF is 40% heme and 60% non-heme iron (Monsen et al., 1978). Estimates of available iron were not made because the only available algorithm for estimating available iron at the time of the study (Monsen & Balintfy, 1982; Monsen et al., 1978) did not incorporate the inhibitory effects of dietary fibre, phytate, calcium or tannins on iron absorption The 3d-FRs and FFQs were then analysed using FP® to calculate the intakes (g/day) of food from major food categories and the iron intakes from these categories. To accomplish this, all the foods on the FFQ and 3d-FR f