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Vitamin B-12 status during pregnancy and infancy : screening tools and assessment of populations at risk… Schroder, Theresa H. 2017

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VITAMIN B-12 STATUS DURING PREGNANCY AND INFANCY: SCREENING TOOLS AND ASSESSMENT OF POPULATIONS AT RISK FOR DEFICIENCY  by  Theresa H Schroder  Vordiplom, University of Konstanz (Germany), 2007 Dipl. LM-Chem., University of Hohenheim (Germany), 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Human Nutrition)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2017  © Theresa H Schroder, 2017 ii  Abstract Low perinatal and infantile vitamin B-12 (B-12) status have been associated with health complications. South Asians and residents of low- and middle-income countries may be at increased risk for low B-12 status.  The overall goal was to facilitate and screen for perinatal, neonatal, and infantile B-12 status. First, a reliable (recovery: 93–98%; CV: <7%) tool for B-12 status assessment in vulnerable populations was developed, using dried blood spot methylmalonic acid (DBS MMA) concentrations. MMA is the most specific functional B-12 biomarker. Given the minimal invasiveness and ease of DBS collection, DBS MMA may be convenient to use in newborns and populations in remote settings to estimate B-12 status. As such, a reference value of elevated neonatal DBS MMA concentration of >29.3 pmol/8-mm punch was computed per clinical guidelines (CLSI EP18-A3c). Further, B-12 status of South Asian and European pregnant women and their newborns living in Vancouver were compared. B-12 status was assessed in 748 healthy Vancouver women (50% South Asian, 50% European) during their 1st and 2nd trimester of pregnancy using multiple B-12 biomarkers, and in their newborns using DBS MMA concentration. South Asian pregnant women had a significantly lower B-12 status than European women, e.g. comparing 1st trimester mean (95% CI) serum total B-12 concentrations [189 (180; 199) pmol/L versus 246 (236; 257) pmol/L; P<0.0001]. This difference in B-12 status was not reflected in the DBS MMA concentrations of their newborns. Last, the prevalence of B-12 deficiency in mothers and their infants living in rural Indonesia was determined. The prevalence of infants (n=221) living in rural Indonesia with serum total B-12 concentrations <191pmol/L followed at age 6-, 9-, and 12-months was 27%. Maternal iii  DBS MMA concentrations at 6 months postpartum were weakly, but statistically significantly (P=0.004), associated with infant serum MMA concentrations. This research suggests DBS MMA is a convenient screening tool with use in vulnerable populations, including newborns. Pregnant South Asian women living in Vancouver and infants living in rural Indonesia were identified as populations at risk for low B-12 status. Future research evaluating outcomes and predictors of low B-12 status is warranted allowing for targeted interventions.  iv  Preface This thesis was prepared according to the requirements for a PhD thesis by The University of British Columbia (UBC) Faculty of Graduate and Postdoctoral Studies. This work is the result of a collaborative effort. All of the work presented henceforth was conducted in the Human Nutrition and Vitamin Metabolism Laboratory in the Faculty of Land and Food Systems at UBC, the Prenatal Screening Laboratory at the British Columbia Children’s Hospital, the Pathology Laboratory at St Paul’s Hospital, Vancouver, Canada, and the Department of Human Nutrition at the University of Otago, New Zealand. Biospecimen and data for the work presented in Chapter 5 were collected at sites in the Sumedang district of West Java, Indonesia; latter biospecimen were processed at the Universitas Padjadjaran, Indonesia. A version of Chapter 2 has been published in the Journal of Nutrition (Schroder TH, Quay TA, Lamers Y. J Nutr. 2014;144:1658-63). Lamers Y designed the overall research project, supervised all activities and conducted part of the method development. Schroder TH developed the analytical method, designed the stability study, conducted specimen collection and preparation, analyzed all data, and wrote the initial draft of the manuscript. Quay TA coordinated the applicability study and conducted most of the specimen collection. Lamers Y was the principal investigator and supervisor on this project and had primary responsibility for the final draft of the manuscript. This project was approved by UBC’s Clinical Research Ethics Board (certificate H11-02269). A version of Chapter 3 has been published in the Journal of Clinical Biochemistry (Schroder TH, Mattman A, Sinclair G, Vallance HD, Lamers Y. Clin Biochem. 2016;49:973-v  8). Schroder TH and Lamers Y developed the research question. Schroder TH drafted the research protocol with consultation from Sinclair G, Vallance HD and Lamers Y. Schroder TH conducted sample and data analysis, and wrote the first draft of the manuscript. Sinclair G and Vallance HD facilitated the sample collection and contributed to manuscript edits. Mattman A contributed to manuscript edits. Lamers Y was the principal investigator on this project and was involved throughout the project in concept formation and manuscript revisions. This project was approved by UBC’s Children’s and Women’s Research Ethics Board (certificate H14-01911). Research outcomes described in Chapter 4 are currently being summarized in the form of two manuscripts, each with the following authors: Schroder TH, Sinclair G, Mattman A, Jung B, Barr SI, Vallance HD, Lamers Y. Schroder TH and Lamers Y developed the main ideas of this research, including the study design. Schroder TH drafted the research protocol in consultation with Sinclair G, Mattman A, Vallance HD, and Lamers Y. Schroder TH was responsible for study coordination and conducted the data analysis and data interpretation, as well as composed the first draft of this chapter. Sample collection and data interpretation were supported by Sinclair G, Mattman A, and Vallance HD; sample collection and analysis were supported by Co-op, Undergraduate Research and Work Learn Students, technicians, and other staff at the Prenatal Screening Laboratory (BC Children’s Hospital) and the Human Nutrition and Vitamin Metabolism Laboratory (UBC); sample analysis was further supported by Jung B and the Pathology Laboratory (BC Children’s Hospital). Barr SI contributed to data analysis, interpretation, and chapter revisions. Lamers Y is the principal investigator of this project, providing oversight and input on all aspects of the study as well as on the chapter composition vi  and revisions. This project has been approved by UBC’s Children’s and Women’s Research Ethics Board (certificate H15-00820). Chapter 5 arose as a result of a secondary analysis from a study designed by Houghton LA, Diana A, and Gibson RS investigating the prevalence and determinants of micronutrient deficiencies and anemia in infants living in the Sumedang District of West Java, Indonesia. Lamers Y established the collaboration with Houghton LA and facilitated this project. Schroder TH and Lamers Y designed the study protocol for this secondary analysis. Schroder TH contributed to identifying a specific research hypothesis, oversaw the biospecimen analysis, and conducted the data analysis and data interpretation, as well as wrote the first draft of this chapter. Houghton LA, Diana A, Gibson RA, and Barr SI provided input on data interpretation and chapter revisions. Biospecimen analyses were supported by Co-op and Undergraduate Research Students at the Human Nutrition and Vitamin Metabolism Laboratory (UBC) as well as staff at the Department of Human Nutrition (University of Otago). Lamers Y was the supervisory investigator of this project and was actively involved throughout the project in concept formation and chapter revisions. This project has been approved by UBC’s Clinical Research Ethics Board (certificate H15-00106).  vii  Table of Contents Abstract ..................................................................................................................................... ii Preface...................................................................................................................................... iv Table of Contents ..................................................................................................................... vi List of Tables ......................................................................................................................... xiii List of Figures ........................................................................................................................ xvi List of Equations ..................................................................................................................... xx List of Abbreviations ............................................................................................................. xxi Acknowledgements .............................................................................................................. xxiii Dedication ............................................................................................................................ xxiv 1. Introduction, Literature Review, and Objectives with Hypotheses ..................................... 1 1.1. Introduction .............................................................................................................. 1 1.2. Review of Literature ................................................................................................. 2 1.2.1. Function of Vitamin B-12 in Fetal and Infantile Development ........................ 2 1.2.2. Assessment of Vitamin B-12 Status in Pregnant Women and Infants ............ 13 1.2.3. Predictors and Prevalence of Vitamin B-12 Deficiency During Pregnancy and Infancy in Canada and World-Wide ....................................................................... 35 1.3. Summary of Rationale ............................................................................................ 44 1.4. Research Objectives and Hypotheses ..................................................................... 46 viii  2. Methylmalonic Acid Quantified in Dried Blood Spots Provides a Precise, Valid, and Stable Measure of Functional Vitamin B-12 Status in Healthy Women ........................... 51 2.1. Summary ................................................................................................................ 51 2.2. Introduction ............................................................................................................ 52 2.3. Materials and Methods ........................................................................................... 54 2.3.1. Reagents and Chemicals .................................................................................. 54 2.3.2. Preparation of Calibrators ............................................................................... 54 2.3.3. Collection of Dried Blood Spot and Plasma Specimens ................................. 55 2.3.4. Sample Processing for Methylmalonic Acid Extraction ................................. 56 2.3.5. Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry System .................................................................................................................... 57 2.3.6. Method Validation Experiments ..................................................................... 58 2.3.7. Testing for Long-term Stability of Methylmalonic Acid Concentrations in Dried Blood Spots .................................................................................................. 60 2.3.8. Statistical Analyses ......................................................................................... 60 2.4. Results .................................................................................................................... 61 2.4.1. Method Development and Optimization ......................................................... 61 2.4.2 Method Validation ........................................................................................... 64 2.4.3 Method Applicability ...................................................................................... 66 2.4.4 Long-term Stability of Methylmalonic Acid in Dried Blood Spots ................ 68 ix  2.5 Discussion .............................................................................................................. 69 2.6 Conclusions ............................................................................................................ 72 3. Reference Interval of Methylmalonic Acid Concentrations in Dried Blood Spots of Healthy, Term Newborns to Facilitate Neonatal Screening of Vitamin B-12 Deficiency . 73 3.1. Summary ................................................................................................................ 73 3.2. Introduction ............................................................................................................ 74 3.3. Materials and Methods ........................................................................................... 76 3.3.1. Study Samples ................................................................................................. 76 3.3.2. Biochemical Analyses ..................................................................................... 77 3.3.3. Statistical Analysis .......................................................................................... 77 3.4. Results .................................................................................................................... 78 3.4.1. Sample Characteristics .................................................................................... 78 3.4.2. Reference Interval ........................................................................................... 80 3.4.3. Impact of Storage Time at Room Temperature on Dried Blood Spot Methylmalonic Acid Concentrations...................................................................... 81 3.4.4. Comparison with Control Samples ................................................................. 83 3.5. Discussion .............................................................................................................. 84 3.6. Conclusions ............................................................................................................ 89 4. Vitamin B-12 Status and Diagnostic Performance of Vitamin B-12 Biomarkers in Pregnant Women of South Asian and European Ethnicity Residing in Vancouver .......... 90 x  4.1. Summary ................................................................................................................ 90 4.2. Introduction ............................................................................................................ 92 4.3. Materials and Methods ........................................................................................... 95 4.3.1. Study Design and Setting ................................................................................ 95 4.3.2. Sample Size ..................................................................................................... 97 4.3.3. Study Sample and Data Collection.................................................................. 98 4.3.4. Biochemical Analyses of Serum Total Vitamin B-12, Holotranscobalamin, Methylmalonic Acid, Total Homocysteine, and Folate ....................................... 100 4.3.5. Classification of Vitamin B-12 and Folate Status ......................................... 103 4.3.6. Statistical Analyses ....................................................................................... 105 4.4. Results .................................................................................................................. 109 4.4.1. Subject Characteristics .................................................................................. 109 4.4.2. Maternal and Neonatal Vitamin B-12 and Folate Status............................... 112 4.4.3. Correlations of Maternal and Neonatal Biomarker Concentrations .............. 119 4.4.4. Receiver Operating Characteristics Curves of Vitamin B-12 Biomarkers to Discriminate Pregnant Women with Impaired Functional Vitamin B-12 Status . 120 4.4.5. Calculation of Methylmalonic Acid-Derived Reference Values for Serum Total Vitamin B-12 and Serum Holotranscobalamin Concentrations .................. 128 4.5. Discussion ............................................................................................................ 129 xi  4.5.1. Prevalence of Vitamin B-12 Deficiency in Pregnant Women of South Asian and European Ethnicity and Their Newborns Residing in Metro Vancouver ...... 130 4.5.2. Comparison of Total Vitamin B-12 and Holotranscobalamin as Indicator of Functional Vitamin B-12 Status in Pregnant Women .......................................... 135 4.5.3. Pregnancy-Specific Reference Values for Total Vitamin B-12 and Holotranscobalamin to Assess Vitamin B-12 Status ............................................ 140 4.5.4. Strengths and Limitations .............................................................................. 142 4.6. Conclusions .......................................................................................................... 145 5. High Prevalence of Vitamin B-12 Deficiency in Rural Indonesian Infants Followed at 6- to 12-Months of Age ........................................................................................................ 147 5.1. Summary .............................................................................................................. 147 5.2. Introduction .......................................................................................................... 148 5.3. Methods ................................................................................................................ 150 5.3.1. Participants and Study Design ....................................................................... 150 5.3.2. Questionnaires and Anthropometric Measures ............................................. 151 5.3.3. Blood Collection ........................................................................................... 151 5.3.4. Biomarker Quantification and Definition of Vitamin B-12 Status ............... 152 5.3.5. Statistical Analyses ....................................................................................... 153 5.4. Results .................................................................................................................. 154 5.4.1. Participant Characteristics ............................................................................. 154 xii  5.4.2. Infant and Maternal Vitamin B-12 Status ..................................................... 155 5.4.3. Predictors of Infantile Vitamin B-12 Status .................................................. 157 5.5. Discussion ............................................................................................................ 160 5.6. Conclusions .......................................................................................................... 165 6. General Discussion, Conclusions and Future Directions ................................................. 166 6.1. Summary of Presented Work ............................................................................... 166 6.2. General Discussion of the Key Findings .............................................................. 169 6.2.1. Dried Blood Spots as a Novel Mean to Assess Vitamin B-12 Status ........... 169 6.2.2. Screening of Vitamin B-12 Status Using Biomarkers .................................. 173 6.2.3. Populations with a High Prevalence of Vitamin B-12 Deficiency ................ 177 6.3. Limitations of the Studies..................................................................................... 183 6.4. Future Directions .................................................................................................. 184 6.4.1. Evaluation of Reference Values and Alternate Indicators for Vitamin B-12 Status in Pregnant Women and Newborns ........................................................... 184 6.4.2. Predictors of Vitamin B-12 Deficiency in Pregnant Women of South Asian Ethnicity in Canada .............................................................................................. 188 6.4.3. Targeted Interventions ................................................................................... 188 Bibliography ......................................................................................................................... 190  xiii  List of Tables Table 1-1: Adverse health outcomes associated with perinatal vitamin B-12 inadequacy as discussed in a systematic literature review by Finkelstein et al. (2016). .................................. 6 Table 1-2: Results of epidemiological studies investigating the relationship between perinatal and infantile vitamin B-12 (B-12) intake or status and infant/child cognition. ...................... 11 Table 1-3: Summary of studies comparing holotranscobalamin (holoTC) and total vitamin B-12 (B-12) in diagnosing B-12 deficiency. ............................................................................... 16 Table 1-4: Characteristics of studies from Figure 1-2. ........................................................... 25 Table 1-5: Select cutoffs and reference values for adult, pregnant and pediatric populations for serum total vitamin B-12 (B-12), methylmalonic acid (MMA), and holotranscobalamin (holoTC) to determine vitamin B-12 deficiency (numbers in brackets indicate references). . 31 Table 1-6: Vitamin B-12 status in Canadian reproductive-aged and pregnant women. ......... 42 Table 2-1: Liquid chromatography conditions and diagram of flow for quantification of methylmalonic acid in dried blood spots. ............................................................................... 57 Table 2-2: Source and compound parameters for API 4000 (AB SCIEX Pte.) for quantification of methylmalonic acid............................................................................................................. 58 Table 2-3: Recovery for the quantitative analysis of methylmalonic acid (MMA) in dried blood spot (DBS) extract................................................................................................................... 66 Table 2-4: Imprecision for the quantitative analysis of methylmalonic acid (MMA) in dried blood spot (DBS) extract and plasma. .................................................................................... 66 xiv  Table 3-1: Characteristics and dried blood spot (DBS) methylmalonic acid (MMA) concentrations in control samples of children treated for inborn errors of metabolism. ........ 84 Table 3-2: Summary of assay performance of different methods for the quantification of methylmalonic acid in newborn dried blood spots using liquid chromatography. ................. 87 Table 4-1: Statistical variables for sample size calculation. ................................................... 98 Table 4-2: Cutoffs and reference values for serum concentrations of total vitamin B-12 (total B-12), holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine (tHcy) used to classify pregnant women’s B-12 status into B-12 deficiency, suboptimal status, and adequacy. ........................................................................................................................ 104 Table 4-3: Subject characteristics [mean (range) or prevalence] of pregnant women and their newborns. .............................................................................................................................. 111 Table 4-4: Mean (95% CI) serum concentrations of total vitamin B-12 (B-12), holotranscobalamin (holoTC), methylmalonic acid (MMA), total homocysteine (tHcy), and folate of women of European and South Asian ethnicity residing in the Lower Mainland (BC, Canada) and prevalence of inadequate and deficient vitamin status during the 1st and 2nd trimester of pregnancy. ......................................................................................................... 113 Table 4-5: Neonatal dried blood spot methylmalonic acid (DBS MMA) concentrations of newborns of women of European and South Asian ethnicity. .............................................. 116 Table 4-6: Spearman’s rank correlation coefficients (ρ) between maternal serum metabolite concentrations at 1st and 2nd trimester and neonatal dried blood spot methylmalonic acid (DBS MMA) concentrations at birth. ............................................................................................. 119 xv  Table 4-7: Coefficients (95% CI) of linear regression models predicting neonatal dried blood spot methylmalonic acid concentrations (natural log-transformed). .................................... 124 Table 5-1: Vitamin B-12 (B-12) status [geometric mean (95% CI)] at 3 study visits and change over time in infants living in the Sumedang District of West Java in rural Indonesia. ........ 157  xvi  List of Figures Figure 1-1: Scheme of the metabolic functions of vitamin B-12 in humans. ........................... 4 Figure 1-2: Median or mean total vitamin B-12 (B-12) concentrations during pregnancy as reported in select studies since 2000, with fitted trend lines and traditional cutoffs (dotted horizontal lines) for B-12 deficiency (<148 pmol/L), suboptimal B-12 status (148-221 pmol/L), and B-12 adequacy (>221 pmol/L). .................................................................. 24 Figure 2-1: Yield of mean (SD) dried blood spot methylmalonic acid (DBS MMA) concentration (n= 10) after varying extraction time and using either extraction buffer [methanol:water (v/v): 5:95] or water. .................................................................................... 61 Figure 2-2: Chromatogram of a dried blood spot extract containing [A]: Succinic acid (SA) and methylmalonic acid (MMA), mass to charge ratio (m/z) of 117→73; and [B]: Internal standard, methyl-d3-malonic acid (d3-MMA), m/z of 120→76. ............................................. 63 Figure 2-3: Linear range for aqueous methylmalonic acid (MMA) standards measured by liquid chromatography-tandem mass spectrometry. ............................................................... 65 Figure 2-4: Scatterplot of plasma methylmalonic acid (MMA) concentrations vs. dried blood spot (DBS) extract MMA concentrations of healthy women (n= 94) indicating a linear relation [(r= 0.97), P< 0.001]. Horizontal line at y= 271 nmol/L represents reference value distinguishing between elevated and normal MMA concentrations. ...................................... 67 Figure 2-5: Differences from baseline in the methylmalonic acid (MMA) concentration of dried blood spot (DBS) extract after storage of DBS at various temperatures and locations for up to 1 year.............................................................................................................................. 68 xvii  Figure 3-1: Tukey box plot of methylmalonic acid in 8-mm neonatal dried blood spots (pmol) with upper and lower limit of the reference interval (solid grey lines) and their 95% confidence interval (dashed grey lines) according to CLSI EP28-A3c (184). .......................................... 79 Figure 3-2: Comparison of different upper and lower limits of reference intervals, computed according to methods proposed by current guidelines CLSI EP28-A3c (184). ...................... 81 Figure 3-3: Correlation between methylmalonic acid in 8-mm dried blood spot (pmol) and time dried blood spot was stored at room temperature (h) and linear prediction with 95% confidence interval. Dashed, vertical line depicts 7 days (168 h). ............................................................ 82 Figure 3-4: Upper and lower limit of the reference intervals computed for methylmalonic acid in dried blood spots (DBSs) stored for <7 days (n= 72) or ≥7 days (n= 88) at room temperature (rt; 18–21°C) using the robust method. .................................................................................. 83 Figure 4-1: Flow diagram of sample collection and analyses, and reasons for missing samples................................................................................................................................................ 110 Figure 4-2: Changes in concentrations (natural log-transformed) of serum total vitamin B-12 (B-12), holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine (tHcy) with gestational age in pregnant women. .................................................................. 118 Figure 4-3: Receiver operating characteristics (ROC) curves for maternal serum total vitamin B-12 (total B-12; black solid line), holotranscobalamin (holoTC; black dashed line), and methylmalonic acid (MMA; grey dotted line) concentrations to discriminate women with A and B (top row): serum MMA >210 nmol/L (and 2-methylcitric acid<MMA); or C and D (bottom row): having a newborn with DBS MMA concentrations >75th percentile. ........... 121 xviii  Figure 4-4: Comparison of (top row): the geometric mean (95% CI) serum methylmalonic acid (MMA) concentrations of pregnant women classified as vitamin B-12 (B-12) deficient and having inadequate B-12 status, respectively, using serum total B-12 concentrations (<148 and <221 pmol/L, respectively) and serum holotranscobalamin (holoTC) concentrations (<35 and <55 pmol/L, respectively); and (bottom row): the geometric mean dried blood spot (DBS) MMA concentrations of their newborns. .............................................................................. 127 Figure 4-5: Plot of maternal serum total vitamin B-12 (B-12) concentration (top row) and maternal serum holotranscobalamin (HoloTC) concentration (bottom row) against maternal serum methylmalonic acid (MMA) concentration (natural log-transformed) with locally weighted smoothing line (LOESS; red line) during the 1st trimester [A; mean (range) gestational age: 11.5 (8.3–13.9) wk; n= 680] and the 2nd trimester [B; mean (range) gestational age: 16.5 (14.9–20.9) wk; n= 702]. ....................................................................................... 129 Figure 5-1: Prevalence (95% CI; Wald estimation) of infants [grey; n: 165 (enrollment), 159 (3-month follow-up), 143 (6-month follow-up)] with vitamin B-12 (B-12) deficiency [serum total B-12 <191 pmol/L (162); dark grey] and inadequate B-12 status (serum total B-12 <220 pmol/L; light grey), and mothers (black; n: 184) with dried blood spot methylmalonic acid concentrations suggestive of functional B-12 deficiency. ............................................ 156 Figure 5-2: Serum total vitamin B-12 (B-12; n= 146) and methylmalonic acid (MMA; n= 115) concentrations by infant age in months (mo). ....................................................................... 158 Figure 5-3: Serum methylmalonic acid (MMA; natural log-transformed) concentration (nmol/L) by serum total vitamin B-12 (B-12) concentration (pmol/L) in infants aged 6 (square; n= 117), 9 (circle; n= 122), and 12 (cross; n= 116) months. ................................................ 159 xix  Figure 5-4: Infant serum methylmalonic acid (MMA; natural log-transformed) concentration (nmol/L) by maternal dried blood spot (DBS) MMA concentration (pmol/8-mm DBS) in infants aged 6 (square), 9 (circle), and 12 (cross) months and their mothers at 6 months postpartum............................................................................................................................. 160  xx  List of Equations Equation 4-1: Sample size calculation. ................................................................................... 97 Equation 4-2: Calculation of the change per week (pmol/L) in maternal serum total vitamin B-12 and holotranscobalamin concentration. ........................................................................... 106 Equation 4-3: Calculation of the mean change per week (%) relative to the 1st trimester concentration in maternal serum total vitamin B-12 and holotranscobalamin concentration................................................................................................................................................ 106 xxi  List of Abbreviations AI adequate intake AIC Aikaike Information Criterion AUC area under the curve B-12 vitamin B-12, cobalamin BC British Columbia c concentration CHMS Canadian Health Measures Survey CI confidence interval CLSI Clinical & Laboratory Standards Institute CV coefficient of variation d day(s) d3-MMA methyl-d3-malonic acid DBS dried blood spot EAR estimated average requirement EDTA ethylenediaminetetraacetic acid GC gas chromatography h hour(s) holoTC holotranscobalamin IQR inter-quartile range LC liquid chromatography LMIC low- and middle-income country MCA 2-methylcitric acid xxii  MMA methylmalonic acid mo month(s) MS mass spectrometry MS/MS tandem mass spectrometry n sample size NHANES National Health and Nutrition Examination Survey NTD neural tube defect OR odds ratio RDA recommended dietary allowance ROC receiver operating characteristics SE standard error SD standard deviation tHcy total homocysteine wk week(s) y year(s) xxiii  Acknowledgements Firstly, I would like to thank my supervisor, Dr. Yvonne Lamers, for providing me with this opportunity, as well as her focused guidance, motivation and tremendous support. None of this work would have been possible without her dedication and commitment to research excellence. I am grateful to have learnt many valuable lessons from her. Danke! I would also like to extend my thanks to my supervisory committee. My deepest gratitude goes to Dr. Susan Barr not only for providing her most constructive feedback on my work but also for her positive energy and encouraging words which kept me motivated; to Dr. Jehannine Austin for asking excellent questions, her enthusiasm, encouragement, and mentorship; and to Dr. Joshua Miller for sharing his expertise within the field and providing me with valuable feedback and insights. My research would not have been possible without Dr. Hilary Vallance, Dr. Graham Sinclair, and Dr. Andre Mattman, whom I would like to thank for their dedication to this work as well as their mentorship and support. I am further indebted to Aly Diana, Dr. Lisa Houghton and Dr. Rosalind Gibson for letting me contribute to an excellent and exciting study as well as to Teo Quay for sharing her research with me. A very special thank you also to Benny, Tom, Tina, Marta, Arianne, Pablo, Ori, and Matthew as well as the many volunteers for their invaluable assistance in my research projects. Further, I would not want to miss thanking all the participants in the studies that were part of my PhD work for their interest, time and commitment. I am grateful to all the grad students, faculty members, and staff in FNH and LFS who helped me on this journey through providing support and companionship; a special thank you to Teo, Fernanda, Kyly, Aviva, Ellie, Alejandra, and Rose for their friendship, encouragement and discussions about research and beyond. I would also like to extend my heartfelt appreciations to Dr. Crystal Karakochuk, Dr. Tim Green, and Dr. Geoffrey Savage for their mentorship, encouragement, and inspiration (in so many ways) throughout my PhD and research career that made me grow academically and personally.  I would like to acknowledge the University of British Columbia for providing trainee funding support, including through the Four Year Doctoral Fellowship (4YF). Lastly, I would like to thank my parents, Ulrike and Herbert, my family and friends near and far, and especially Omid, for supporting this endeavour of mine and believing in me.  xxiv  Dedication   Für Opa und für Sophia und ihre Mama und Freundschaft, die trotz aller Ferne hält.1  1. Introduction, Literature Review, and Objectives with Hypotheses 1.1.Introduction Once referred to as “nature’s most beautiful cofactor” (1), the utility of vitamin B-12 (B-12) to treat deficiencies1 and improve health of humans was discovered more than 100 years ago and has been studied ever since (2). The discovery of B-12 was subject of 3 Nobel Prizes: to G. Whipple, G. Minot, and W. Murphy in 1934 for treating pernicious anemia — what was later the clinical hallmark for B-12 deficiency; and to D. Hodgkin in 1964 and to R.B. Woodward in 1965 for the characterization and synthesis, respectively, of B-12. Today, symptoms of clinical B-12 deficiency and the biochemical function of B-12 are well elucidated (3,4). Current research focuses on identifying the role of subtle B-12 deficiency in adverse health outcomes, finding consensus on the definition and assessment of stages of B-12 depletion in various populations, and the prevention of B-12 inadequacy world-wide (5–8).  Pregnancy and infancy are important stages of rapid growth and development (9,10). Nutrient adequacy is especially crucial during these early stages of life for healthy fetal and infant development and long-term health (11). Valid and reliable biomarkers can provide insight into the nutrient status of pregnant women and infants (8,12). Developing and validating tools and biomarkers for maternal, neonatal and infantile B-12 status assessment will facilitate screening for B-12 inadequacy in these vulnerable populations. Identifying populations or individuals with B-12 inadequacy will allow for targeted interventions to treat and prevent B-12 inadequacy during these important stages of development.                                                  1 For the purpose of this work, B-12 status is denoted as deficiency, suboptimal status, and adequacy; inadequacy refers to combined suboptimal status and deficiency. Biomarkers and cutoffs are discussed in Chapter 1.2.2.  2  1.2.Review of Literature The aim of this review is to summarize the current state of knowledge on B-12 status, assessment, and prevalence of deficiency2 during pregnancy and infancy. The associations between perinatal and infantile B-12 deficiency with maternal health and infant development are described to illustrate the importance of B-12 adequacy during these stages of rapid growth. Further, special attention is given to the current methods of assessing B-12 status as well as the definition of B-12 deficiency and suboptimal B-12 status in pregnant women and infants. Predictors of maternal and infant B-12 status are explored. Populations with potentially increased risk for a high prevalence of B-12 deficiency3 are identified. The overall goal is to justify research performed on facilitating and applying screening for B-12 deficiency and suboptimal B-12 status during pregnancy and infancy in vulnerable populations. 1.2.1. Function of Vitamin B-12 in Fetal and Infantile Development Biochemistry of vitamin B-12  The scientific use of the term “vitamin B-12” describes a cyano-, hydroxo-, 5’-deoxyadenosyl-, or methyl-group linked to a cob(III)alamin moiety (4,13). 5’-deoxyadenosylcobalamin and methylcobalamin are the 2 metabolically active forms of B-12 (13,14). Hydroxocobalamin and cyanocobalamin are forms of B-12 found in fortified foods or supplements and can be converted into metabolically active forms (4).                                                   2 For the purpose of this work, B-12 status is denoted as deficiency, suboptimal status, and adequacy; inadequacy refers to combined suboptimal status and deficiency. Biomarkers and cutoffs are discussed in Chapter 1.2.2. 3 The prevalence of B-12 deficiency (total B-12 <148 pmol/L) among people (aged ≥3 y) residing in the US is 5% (84). Assuming that the US represents a healthy population regarding B-12 status (5), for the purpose of this work, a prevalence of B-12 deficiency of more than 5% in a population will be considered high. 3  In human metabolism, B-12 is involved in 2 main reactions (9,10; Figure 1-1). In the cytosol, methionine synthase requires methylcobalamin as a cofactor to catalyze the methylation of homocysteine to methionine as part of one-carbon metabolism. In this reaction, a methyl group is transferred from 5-methyltetrahydrofolate to homocysteine rendering tetrahydrofolate and methionine. The latter is the precursor for S-adenosylmethionine, the universal methyl donor. S-adenosylmethionine is required for essential reactions, including the methylation of phospholipids, neurotransmitters, amines, DNA, RNA, histones, and myelin basic protein (13). B-12 deficiency impairs the regeneration of methionine resulting in decreased formation of S-adenosylmethionine, increased concentrations of homocysteine, and the trapping of folates as 5-methyltetrahydrofolate (3). Clinical B-12 deficiency due to pernicious anemia, a disease leading to malabsorption of B-12, has been associated with elevated circulating total homocysteine (tHcy) concentrations (15) and tHcy is a functional biomarker of B-12 status. However, given the requirement of multiple B-vitamins in the homocysteine catabolism, tHcy concentrations are determined by intake and status of multiple B-vitamins, including folate status (16,17). Elevated circulating maternal tHcy concentrations have been associated with several adverse pregnancy outcomes, independent of B-12 status (18,19). Taken together, B-12 participates in one-carbon metabolism and, thus, is required for essential methylation reactions and homocysteine remethylation. 4   Figure 1-1: Scheme of the metabolic functions of vitamin B-12 in humans.  Figure is based on publications by Hannibal et al. and Watkins and Rosenblatt (8,20). PLP: Pyridoxal-5’-phosphate (coenzyme form of vitamin B6); CBS: Cystathionine-β-synthase; MTHF: 5-Methyltetrahydrofolate; MTHFR: 5,10-Methylenetetrahydrofolate reductase; MeTHF: 5,10-Methylenetetrahydrofolate; SAH: S-Adenosylhomocysteine; BHMT: Betainehydroxymethyltransferase; MS: Methionine synthase; SAM: S-Adenosylmethionine; DMG: Dimethylglycine; THF: Tetrahydrofolate; MeCbl: Methylcobalamin; Cbl: Cobalamin; TC-Cbl: Holotranscobalamin; FA: Fatty acid; TCA: Tricarboxylic acid; BCAA: Branched-chain amino acid; AdoCbl: 5’-Deoxy-5’-adenosylcobalamin; MMCM: Methylmalonyl-CoA mutase.  In the mitochondria, 5’-deoxyadenosylcobalamin is involved in the interconversion of methylmalonyl-CoA and its stereoisomer succinyl-CoA. The reaction is part of odd-chain fatty acid and branched-chain amino acid metabolism (13). In the case of intracellular B-12 deficiency, excess methylmalonyl-CoA is metabolized into methylmalonic acid (MMA) 5  leading to elevated circulating MMA concentrations, another functional biomarker of B-12 status (17,21,22).  The metabolic functions of B-12 in one-carbon and odd-chain fatty acid metabolism also highlight its importance during rapid stages of growth and neurocognitive development, i.e. fetal and infant development. The involvement of B-12 in one-carbon metabolism has been suggested to make the vitamin essential for cell synthesis and mechanisms of epigenetic regulation (23,24). Cell synthesis is most active during fetal and infant development. Critical periods of epigenetic regulation occur in utero (25). Additionally, B-12 is hypothesized to be involved in myelin formation (26–28), which surrounds the axons of nerve sheaths and is crucial for neural function. Myelination of the brain (and cognitive development) is most active in the first 2 years of life (27). It has been suggested that intracellular B-12 deficiency during important and rapid stages of growth may lead to a wide array of adverse health outcomes, some of which are discussed below.  Vitamin B-12 status and perinatal and infant health Prolonged perinatal and infantile B-12 inadequacies have been associated with several adverse health outcomes in mother and offspring, which have been subject of multiple reviews (7,29–34). Finkelstein et al. (2016) have recently discussed adverse health outcomes inversely associated with perinatal B-12 status in a systematic literature review (7). The authors identified 123 human studies (case reports excluded) focussing on the association between B-12 status or intake of pregnant women and outcomes in the newborn and infant. The results are summarized in Table 1-1. Most evidence, however, is based on cross-sectional, case-control, and cohort studies. Only 1 randomized placebo-controlled trial could be identified by 6  the authors. The trial included 366 mother-infant dyads in India with a prevalence of 51% maternal B-12 deficiency (serum total B-12 <150 pmol/L) at enrolment (35). Oral supplementation with 50 µg/d B-12 during pregnancy [Recommended Dietary Allowance (RDA): 2.6 µg/d] significantly increased maternal serum, infant serum and breastmilk B-12 concentrations and lowered the incidence of intra-uterine growth restriction (25% in treatment vs. 34% in placebo group). Despite the need for more comprehensive, prospective and randomized trials, the wide array and severity of adverse health consequences in mother and offspring associated with B-12 inadequacy are recognized and emphasize the need and importance of B-12 adequacy during pregnancy. Table 1-1: Adverse health outcomes associated with perinatal vitamin B-12 inadequacy as discussed in a systematic literature review by Finkelstein et al. (2016)1. Adverse health outcomes associated with perinatal vitamin B-12 inadequacy Maternal anemia  Neural tube defects and other congenital anomalies Preeclampsia Infant/child vitamin B-12 deficiency Gestational diabetes Impaired infant cognitive development Spontaneous abortion Infant/child insulin resistance Low birth weight Excessive crying in the infant 1 Reference: (7)  There is strong evidence for the inverse association between maternal B-12 status and occurrence of neural tube defects (NTDs). Maternal B-12 inadequacy has been identified as an independent risk factor for having a pregnancy affected by an NTD (36,37). Case-control studies found that women with NTD-affected pregnancies had a significantly lower B-12 status, as assessed by total B-12, holotranscobalamin (holoTC), and MMA concentrations, than controls (36–39). In 2007, a population-based case-control study conducted in 89 NTD cases (stillborn or alive) and 422 controls in Ontario, Canada, estimated that 34% of NTDs in Canada may be due to maternal B-12 deficiency (holoTC <55 pmol/L) (38). Additionally, a daily 7  maternal B-12 intake below 2.1 μg during pregnancy has been associated with an increased risk of having a newborn with coronary heart defects [OR (95% CI): 1.86 (1.03; 3.35)] in a Dutch case-control study (n: 192 cases, 216 controls) (40). Yet, no association between serum total B-12 concentration and occurrence of congenital heart defects was observed in a case-control study (n: 140 cases, 280 controls) in California (41). Further, while 1 case-control study (n: 96 cases, 88 controls) reported an association between maternal B-12 deficiency (total B-12 <185 pmol/L) and orofacial clefts [OR (95% CI): 3.1 (1.7; 7.4)] (42), a recent meta-analysis of 7 studies did not confirm these findings [Cohen’s d (95% CI): -0.01 (-0.22; 0.18)] (43). The prevalence of congenital anomalies in live births was 373 per 10,000 births (excluding Down Syndrome) in Canada in 2009 (44). Improving maternal B-12 status, especially during the time of neural tube closure (28 d of gestation) and early embryonic development, may have the potential to decrease the occurrence of NTDs and other congenital anomalies. Women of South Asian ethnicity have a higher prevalence of NTD-affected pregnancies compared to women of European or other ethnicities. An overall incidence of 4.1 (95% CI: 3.1; 5.4) and 4.5 (95% CI: 4.2; 4.9) NTD cases per 1,000 alive or still births was reported for India in 2 independent systematic reviews in 2013 and 2015, respectively, despite folic acid fortification and supplementation programs (45,46). A population-based study in Birmingham, UK, revealed that women of South Asian ethnicity also had a significantly higher rate of pregnancies affected by any congenital anomalies at 32.2 per 1,000 alive or still births (1,689/53,832), than women of European ethnicity at 27.5 per 1,000 alive or still births (6690/318,000) from 1960–84 (47). South Asians are Canada’s largest ethnic minority (48) and may be at an increased risk for pregnancies affected by NTDs. While NTDs have been 8  associated with maternal B-12 deficiency, it is not known whether the increased NTD risk for South Asian women is related to their B-12 status.  South Asian infants have been described as having a lower birth weight and a relatively higher body fat (49–51), an anthropometry that may predispose to an increased risk for cardiometabolic disease (52), compared to European infants or infants of other ethnicities. A recent study, utilizing data from 2 prospective Canadian birth cohorts (FAMILY and START; n: 401 South Asian, 389 European) reported a significantly lower birth weight [mean (SE): 3283 (22) g vs. 3517 (22) g; P< 0.0001] and higher triceps and subscapular skinfold thickness [mean sum (SE): 11.7 (0.1) mm vs. 10.6 (0.1) mm; P< 0.0001] in newborns of South Asian compared to European parents (49). Lower birth weight has been associated with an accelerated weight gain and higher systolic blood pressure at 12 mo of age, both risk factors for cardiometabolic disease later in life (53), in a study of 189 and 371 British-born infants of South Asian and European ethnicity, respectively (52). Interestingly, maternal B-12 status has been associated with measures of fetal and infant anthropometry (35,51,54–56) as well as offspring cardiometabolic health in several studies of South Asian populations (57–59). It has been described that maternal supplementation with 50 μg B-12/d led to a lower incidence of intra-uterine growth restriction in a randomized-controlled trial of 366 women residing in Bangalore, India (n: 33/131 vs. 43/125) (35). Yet the difference in intra-uterine growth restriction did not reach statistical significance (P= 0.11) due to insufficient power. Further, a recent systematic review and meta-analysis of 22 studies (n= 11,993) reported a higher risk for low birth weight [<2500 g; adjusted risk ratio (95% CI): 1.15 (1.01; 1.31)] and preterm birth [<37 wk; adjusted risk ratio (95% CI): 1.21 (0.99; 1.49)] among women of all ethnicities with B-12 deficiency (total B-12 <148 pmol/L) during pregnancy (5– >37 wk of gestation) (60). 9  Further, an association between maternal B-12 status and birth weight [change (95% CI) in birth weight per 1 SD increase in B-12 concentration: 22.2 (2.1; 42.4) g] was observed in studies conducted in low- and middle-income countries (LMIC); 80% of studies (4 of 5; total n= 1995) had been conducted in India (60). Maternal B-12 deficiency (total B-12 <148 pmol/L) during early pregnancy (~11 wk of gestation) has also been associated with a 27% increase in insulin resistance using the homeostasis model assessment (HOMA-IR) in the offspring at 6–8 y of age in a study of 541 mother-infant dyads in Nepal (57). Similar findings were reported from a study of 700 mother-infant dyads in Pune, India, in which maternal total B-12 concentration at 18 wk of gestation was significantly (P= 0.03) inversely associated with offspring insulin resistance, also measured by using HOMA-IR) at 6 y of age (59). As such, South Asian infants have been reported to be at an increased risk for impaired cardiometabolic health, which may partially be attributed to maternal B-12 deficiency. In addition to pregnancy-related adverse health outcomes, maternal B-12 deficiency can also lead to neonatal and infantile B-12 deficiency. Infant B-12 deficiency typically occurs second to maternal B-12 deficiency (32,61,62). Infants born to and breastfed by B-12 depleted mothers, e.g., vegetarian or vegan mothers, are at very high risk of developing progressive neurological disorders at 4 to 12 months of age, as described in case reports (27,28,32,62,63). In Canada, Roumeliotis et al. (2012) reported 2 cases of infants aged 7 and 9 months (mo) who presented with anemia, failure to thrive, hypotonia, lethargy, and other symptoms (63). The mothers of both infants were diagnosed with B-12 deficiency (serum total B-12 <107 pmol/L) due to a life-long B-12 deficient diet (strictly vegan) and pernicious anemia, respectively. Neonatal and infantile B-12 deficiency typically present with a variety of non-specific symptoms (32,62) such as those described by Roumeliotis et al. (2012) (63). It is therefore 10  often missed or misdiagnosed (64). Multiple clinicians have suggested that neonatal B-12 deficiency is considerably under-diagnosed (8,65–68). Diagnosis and treatment of infantile B-12 deficiency is important to prevent more severe long-term consequences (69), such as persistent neurocognitive damage (discussed below). Hence, screening for neonatal B-12 deficiency using a population-based approach — especially in at-risk populations — has been proposed (62,64,67). However, convenient assessment tools suitable for B-12 deficiency screening in newborns, infants, and large populations are lacking. B-12 deficiency in early life has long-term health consequences. The cause of impaired neurocognitive development through perinatal and infantile B-12 deficiency has been the subject of several narrative reviews (32,33,70). B-12 is involved in neuron myelination and brain development (26,27). Brain growth is very rapid during the first 2 years of life (27) suggesting that infants may be most vulnerable to B-12 deficiency and related adverse effects on cognitive development. Infant B-12 deficiency has been associated with demyelination and brain atrophy accompanied by symptoms of delayed motor development and impaired neurocognitive function in several case studies (26,27,63) and 1 review of case studies (32). Perinatal or infantile B-12 status and cognitive function in the infant was also the subject of a limited number of epidemiological studies (71–79). The results of the studies are summarized in Table 1-2. There appears to be some evidence for a positive association between infant/child B-12 status and infant/child cognitive function, especially in infants with tHcy (>6.5 µmol/L) or total B-12 (median: ~260 pmol/L) concentrations indicative of B-12 inadequacy (73,74,76,77). Recently, infant B-12 status assessed by MMA and tHcy concentrations, but not total B-12 concentrations, at age 2–12 mo has been associated with cognitive function at age 5 y in a prospective trial of 320 Nepalese children (79). The findings associating maternal 11  perinatal B-12 status or intake and offspring cognition are inconsistent. Srinivasan et al. (2016) conducted a randomized, placebo-controlled trial in 366 pregnant women (<14 wk gestation), who received oral B-12 supplementation (50 µg/d) or placebo throughout pregnancy and until 6-wk postpartum (80); 50% of participants were B-12 deficient (total B-12 <150 pmol/L) at baseline. There was no difference between treatment and placebo group in the Bayley Scales of Infant Development-III scores in the 9-mo old infants (n= 178). Yet, maternal perinatal tHcy concentrations were a significant predictor of the Bayley subscales expressive language and fine motor skills, but not receptive language or gross motor skills. In summary, there appears to be evidence that infant B-12 status is positively associated with cognitive development; however, findings of perinatal B-12 status or intake and offspring cognitive development remain inconclusive.  Table 1-2: Results of epidemiological studies investigating the relationship between perinatal and infantile vitamin B-12 (B-12) intake or status and infant/child cognition.1 Independent variable Dependent variable Sample size Outcome Ref. Maternal 1st trimester B-12 intake (FFQ) Child (9 y) cognition (BSID-III) 253 B-12 intake <2 µg/d was associated with decreased child mental, not psychomotor, development. (71) Maternal pregnancy B-12 intake (FFQ) Child (8 y) IQ 6,019 Positive association between maternal B-12 intake and child IQ, attenuated after adjusting for confounders. (72) Maternal B-12 status at 16 wk and 36 wk gestation Child (18 mo) development (BSID-III) 154 No significant correlation. (81) Maternal 3rd trimester B-12 status Child (9–10 y) cognition (multiple tests incl. Kaufman) 536 No association between maternal B-12 status and any test of cognition. (75) 12  Independent variable Dependent variable Sample size Outcome Ref. Maternal B-12 status at 28 wk gestation Child (9 y) cognition (multiple tests) 49 +592 Children of mothers in the lowest compared to highest B-12 decile performed worse in 2/4 tests. (78) Pregnant women (<14 wk) receiving 50 µg B-12/d Infant (9 mo) cognition (BSID-III) 183+1832 No difference between groups. (75) Infant B-12 status at 1.5, 4, and 6 mo Infant cognition (AIMS, ASQ) 80, 68, 66 Breastfeeding was associated with lower B-12 status and lower cognition. (73) Infant B-12 status at 2–12 mo Child (5 y) cognition  (ASQ, NEPSY) 320 MMA, tHcy, and cB-12 was associated with cognition, not total B-12 (79) B-12 deficient (tHcy >6.5 µmol/L) infants (6 mo) receiving 400 µg B-12 i.m. Infant (7 mo) cognition (AIMS, ASQ) 16+162 B-12 injections resulted in significantly higher increase in infant cognitive function compared to control. (73) B-12 deficient (tHcy >6.5 µmol/L) infants (<8 mo) receiving 400 µg B-12 i.m. Infant cognition (AIMS) 1 mo after treatment 42+372 B-12 injections resulted in significantly higher increase in infant cognition compared to control. (77) Children (12–18 mo) Child cognition (BSID-III) 145 B-12 status was positively associated with mental, not psychomotor, development. (76) Children (6–30 mo) receiving 1.8 µg B-12/d Child cognition (ASQ) 6 mo after start of treatment 220+2202 B-12 supplementation resulted in a significant improvement in gross motor function only. (74) 1 FFQ: food frequency questionnaire; y: year(s); d: day(s); wk: week(s); mo: month(s); BSID-III: Bayley Scales of Infant Development-III; AIMS: Alberta Infants Motor Scale; ASQ: Ages and Stages Questionnaire; NEPSY: Developmental Neuropsychological Assessment; MMA: methylmalonic acid; tHcy: total homocysteine; cB-12: combined indicator of B-12 status; i.m.: intramuscular.  2 Intervention/cases + controls.  13   Given its role in the one-carbon metabolism and odd chain fatty acid metabolism, B-12 is involved in biological processes which are most active during rapid stages of growth, such as pregnancy and infancy. Impaired B-12 function during those crucial stages of development can lead to life-long impairment. Perinatal and infantile B-12 deficiency has been associated with various adverse health outcomes, including NTDs, anemia, and possibly impaired neurocognitive development. While it has been shown that some symptoms of B-12 deficiency in newborns and infants can be treated by B-12 injections, neurocognitive impairment may persist despite treatment (27). Hence, B-12 adequacy during pregnancy and infancy is crucial for long-term health in the offspring and child, respectively. This leads to the need for valid and convenient methods which allow for screening and testing of B-12 status in pregnant women, newborns, and infants as well as identifying populations at risk for deficiency. 1.2.2. Assessment of Vitamin B-12 Status in Pregnant Women and Infants Strengths and limitations of biomarkers for vitamin B-12 status  The clinical hallmarks for defining B-12 deficiency were historically macrocytic or megaloblastic anemia and neurologic dysfunction (15,82). The often non-specific symptoms of early B-12 deficiency and the potential development of severe neurological damage without the presence of anemia (83) highlight the importance of sensitive diagnostic tests for B-12 deficiency. Today, analytical methods are available for 4 different biomarkers for B-12 status assessment in all populations (84,85). The 2 direct biomarkers are total cobalamin, or total B-12, and holoTC; the 2 functional biomarkers are MMA and tHcy.  14  The measurement of total B-12 in serum was the first biochemical test available for B-12 status assessment. However, serum/plasma total B-12 has been criticised for being a non-sensitive indicator for B-12 status (8). In circulation, 60% to 80% of B-12 is bound to haptocorrin (86). In a study of 27 healthy pregnant women, the fraction of total B-12 bound to haptocorrin ranged from 70% to 80% throughout pregnancy (13–36 wk of gestation) (87). The function of haptocorrin or haptocorrin-bound B-12 is not yet understood. It is believed that haptocorrin-bound B-12 is unavailable to tissues (88). The high fraction of circulating B-12 being unavailable to tissues may, in part, explain the low sensitivity of total B-12 as a biomarker of B-12 status. Additionally, age and pregnancy-related physiological changes have been identified to confound total B-12 concentrations and are discussed below. Total B-12 can be quantified in human serum or plasma by various methods, including microbiological and radio-assays (89–92). Caution may be warranted when comparing data generated by different methods. Discrepancies of up to 54% (DxI 800 Unicel versus Architect i2000sr) in total B-12 concentrations of samples from 96 healthy adults have been reported when comparing modern fully automated chemiluminescence immunoassay analyzers (Abbott Architect i2000sr, Beckmann-Coulter Access, Bayer Advia Centaur, Roche Elecsys, Beckman-Coulter DxI 800 Unicel, Roche Cobas E 601) (90,93,94). Despite these limitations, serum total B-12 is the most commonly used biomarker for B-12 status in all populations, including infants and pregnant women. Holotranscobalamin is the alternate direct indicator for B-12 status assessment. The fraction of B-12 in circulation which is not bound to haptocorrin is bound to transcobalamin (87,95,96). HoloTC (B-12 bound to transcobalamin) carries on average 10% to 30% of total B-12 (87,95). It is readily taken up into tissue cells by a highly specific receptor, CD320 (88). 15  The receptor can be found in all tissues, including the human placenta (97). The presence of CD320 in placental tissue cells with findings from radioactive isotope tracer studies in rabbits (98) led to the hypothesis that holoTC is the primary B-12 form which can be transported across the placenta (10). Given this biological function, holoTC has been hypothesized to provide a more sensitive and specific measure of B-12 status than total B-12 in all populations, including pregnant women (86,95). Studies comparing the performance of holoTC and total B-12 to diagnose individuals with B-12 deficiency are summarized in Table 1-3. It appears that most studies identified holoTC as the significantly better diagnostic tool. Studies with the smallest sample sizes (n: 195–607), i.e. 2 studies in predominantly vegetarian and vegan adults (88% and 54% of the study population, respectively) (99,100) and 1 study of older adults (≥60 y) (101), failed to detect a difference, which might be explained by insufficient statistical power (102). All studies reported significantly lower holoTC and total B-12 concentrations in the B-12 deficient group compared to the non-deficient group, indicating specificity of holoTC and total B-12 as B-12 biomarker. The diagnostic performance of holoTC to discriminate pregnant women with or without functional B-12 deficiency has not yet been determined and awaits further investigation.  16  Table 1-3: Summary of studies comparing holotranscobalamin (holoTC) and total vitamin B-12 (B-12) in diagnosing B-12 deficiency.1 Study population No of subjects (with B-12 deficiency) Definition of B-12 deficiency AUC HoloTC2 AUC  B-122 Ref. Outpatients (8–92 y) 758 (174) MMA ≥300 nmol/L (4th quartile) 0.71 0.60 (103) Omnivorous and vegetarian adults (21–73 y) 204 (68) MMA >271 nmol/L 0.88 0.84 (100) Omnivorous and vegan men (23–78 y) 195 (56) MMA >750 nmol/L,  tHcy >15 μmol/L 0.87 0.86 (99) Adults (≥18 y) with suspected B-12 deficiency (MMA >280 nmol/L in past 4 y) 806 (24) MMA >750 nmol/L, tHcy >15 μmol/L 0.90 0.85 (104) Older adults (≥65 y) with normal renal function 607 (37) MMA >350 nmol/L tHcy >13 μmol/L 0.82 0.83 (101) Older adults (≥65 y)  1651 (70) MMA >750 nmol/L 0.85 0.76 (102) Older adults (≥65 y)  1651 (129) MMA >450 nmol/L 0.80 0.73 (102) Older adults (63–97 y) 699 (67) RBC cobalamin <33 pmol/L (2.5th percentile of 120 healthy adults) 0.90 0.80 (105) 1 No: number; AUC: area under the curve (here: obtained after receiver operating curve analysis); ref: reference; y: years of age; RBC: red blood cell. 2 bold numbers indicate significant difference between holoTC and total B-12.   Some confounders of holoTC concentrations have been identified, including sex, age (women: ρ= 0.31), renal function (men: ρ= 0.13), genetic polymorphisms (TCN2 766C>G) and sample preparation (86). The use of EDTA as anticoagulant in sample collection has been reported to have a 140% positive bias compared to other anticoagulants (106). Confounding through different analytical methods ranges within 5% to 20% (86,106–108). As such, the effect of different analytical methods is estimated to be lower on holoTC concentrations than 17  total B-12 concentrations. However, both direct B-12 biomarkers have analytical and non-analytical confounding factors, including age and physiological stage (e.g. pregnancy), that highlight the limitations of each biomarker.  Vitamin B-12 is required for the conversion of methylmalonyl-CoA to succinyl-CoA . Excess methylmalonyl-CoA is metabolized to MMA, which is released from the cells. Circulating MMA concentration has been described as the most specific functional indicator of B-12 status (22,109). Circulating MMA concentrations are elevated in individuals with B-12 deficiency (defined as serum total B-12 <148 pmol/L in combination with hematological and neurological abnormalities) (22), including infants (21,62,110). MMA as a sensitive and specific biomarker of B-12 status in infants is discussed in detail below. Elevated MMA concentration is commonly used as an indicator of impaired B-12 function (100–103,111). Elevated plasma/serum or urinary MMA concentrations are also referred to as functional B-12 deficiency. MMA is a dicarboxylic acid and can be quantified by the stable isotope-dilution technique using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (112,113). This is a precise and reliable method for the quantification of small molecules in biological samples. Methylmalonic acid concentration is affected by impaired renal function and glomerular filtration rate as well as age and potentially by gut microbiota (114). While sex explained 0.2% of the variation in MMA concentrations in adults aged 47–49 y (n= 3,684) (115), no difference by sex was observed in MMA concentrations of infants and children aged 4 d to 19 y (n= 700) (116). Age has been identified as a predictor of plasma MMA concentrations, especially in infants where an increase in MMA concentrations was observed between 0–6 mo followed by a decrease to adult levels by 12 mo (116). As such, when 18  assessing MMA concentrations as indicator of infantile B-12 status, age needs to be accounted for. Impaired renal function is mostly discussed as a confounder of elevated MMA concentrations in older adults (117). Also pregnancy is accompanied by changes in renal function. During pregnancy, renal clearance increases with increased blood flow and glomerular filtration rate (118) which may in turn decrease MMA concentrations. Quantification of 2-methylcitric acid (MCA; see Figure 1-1) in addition to MMA may control for altered renal function (16,117,119,120). MCA is cleared by the kidney at a similar rate as creatinine, the gold standard for assessment of renal function (119). In patients with renal insufficiency (n= 15), MCA concentrations in serum and cerebrospinal fluid were significantly higher than MMA concentrations (119); matched controls with sufficient renal function had MCA < MMA concentrations, regardless of their B-12 status. Thus, if serum MCA > MMA concentrations, MMA concentrations may be increased due to renal insufficiency rather than B-12 deficiency (119,121). MCA and MMA can be quantified simultaneously in biological samples using LC-MS/MS (122). Although serum creatinine is the gold standard to control for renal function (114), MCA:MMA ratio can serve in identifying cases with elevated MMA concentrations due to renal insufficiency rather than B-12 deficiency.   It has further been argued that MMA may have some limitations as specific indicator of B-12 status in pregnant women. In a study of 75 pregnant women, MMA concentrations were not correlated with total B-12 concentrations and were elevated in 30% of the study population independent of their total B-12 concentrations (123). The HIBCH genotype has been found to have a significant effect (P< 0.0001) on MMA concentrations throughout pregnancy (14–34 gestational wk) independent of total B-12 concentrations (124) and may explain some of the observed discrepancy between MMA and total B-12 concentrations in 19  pregnant women. In a study of 184 healthy pregnant women, CC homozygotes (37.5%) compared to TT homozygotes (19.0%) of the HIBCH polymorphism had significantly higher MMA concentrations [mean (range): e.g. 150 (130–250) nmol/L vs. 100 (80–140) nmol/L at 14 wk of gestation] while total B-12 concentrations did not differ by genotype. A similar, yet more pronounced effect was reported in cord blood samples (n= 110) (124). CC homozygotes had almost twice the MMA concentrations of TT homozygotes [mean (range): 470 (300–670) nmol/L vs. 260 (230–300) nmol/L). However, this effect as well as a poor correlation between total B-12 and MMA has yet only been shown in single studies. More research is needed to elucidate the relationship between B-12 biomarker concentrations in pregnant women, their newborns, and infants to understand the limitations of each biomarker. Plasma tHcy is a second functional biomarker of B-12 status, and inadequate B-12 status is associated with elevated plasma tHcy concentration (117,125). Plasma tHcy concentration has low specificity as a B-12 biomarker because it is strongly influenced by folate and other B-vitamin concentrations (16,17). The strongest determinant of circulating tHcy concentration is folate status (16,17,22). Experts do not recommend the use of tHcy as a sole indicator of B-12 status in adults (8,126).  None of the 4 B-12 biomarkers is considered the ‘gold standard’ for B-12 status assessment. Algorithms and mathematical models including multiple biomarker and factors have been suggested to provide advantages in assessing B-12 status (8). Fedosov et al. (2015) recently proposed a mathematical model combining multiple B-12 biomarkers (127). The model distinguishes among 5 categories of B-12 status ranging from ‘probable B-12 deficiency’ to ‘elevated B-12’ (127). Additionally, it includes folate status, sex, and age, to control for confounding by these factors. It was shown to be a sensitive indicator of B-12 status 20  in adults with cognitive impairment (128) and anemia (129) due to B-12 deficiency. However, the model requires the quantification of at least 2 biomarkers (127), which makes its use in clinical settings challenging where commonly only 1 direct biomarker is quantified by fully automated assays. Additionally, the model has today only been assessed in adults and older adults, not in pregnant women or infants.  Experts involved in the National Health And Nutrition Examination Survey (NHANES) in the United States recommended the measurement of 1 direct and 1 functional biomarker (126,130). Classification of adults’ B-12 status by only 1 biomarker (total B-12 ≤148 pmol/L or MMA >376 nmol/L) compared to 2 biomarkers (total B-12 ≤148 pmol/L and MMA >376 nmol/L) led to a discrepancy of up to 88% in the NHANES 1999–2004, i.e. only 22% of the individuals with total B-12 concentrations ≤148 pmol/L had elevated MMA concentrations (>376 nmol/L) (130). The discrepancy was suggested to be related to genetic influences on biomarker concentrations or milder forms of B-12 depletion leading to changes in 1 but not the other biomarker. B-12 status assessment in the NHANES currently employs a combination of serum total B-12 and MMA (84).  Dried blood spot methylmalonic acid concentrations  Since the 1970s, newborn screening programs have been implemented in many countries, including Canada, for early diagnosis of inborn errors of metabolism (131,132). As part of the program, dried blood spot (DBS) samples are collected from newborns within 24–48 h after birth (131). Metabolites are quantified in DBSs using LC-MS/MS. Several inborn errors of B-12 metabolism are known and detected in the newborn screening programs. Mutations in the genes encoding methylmalonyl-CoA mutase (MMCM) or other enzymes 21  involved in intracellular B-12 trafficking (see Figure 1-1), cause mild to severe elevations in circulating MMA concentrations, referred to as methylmalonic acidemia (serum MMA: 0.3 to >1000 µmol/L) (122,133,134). Cases of methylmalonic acidemia are diagnosed in the newborn screening programs after an initial screen, i.e., 1st-tier testing, for abnormal propionylcarnitine or methionine levels in the DBS samples (122,135). This screening test is followed by 2nd-tier analyses to confirm methylmalonic acidemia, such as assessment of tHcy or MMA concentrations in neonatal venous blood samples or functional studies in the newborn (8,20). In most programs, 2nd-tier analyses involve an elaborate 2nd clinic visit including blood draws or biopsies from the newborn (8). This can impose stress and anxiety on the families (136). Some programs utilize methods quantifying MMA in residual neonatal DBSs (122,135,137). However, the methods are not sensitive enough to identify newborns with mild methylmalonic acidemia related to mild inborn errors of B-12 metabolism (135,137) or nutritional B-12 deficiency (64). In addition to newborn screening, DBSs are a convenient collection tool for biological samples with increasing interest and application in epidemiological studies (138–141). DBS collection compared to traditional methods that include venipuncture has the advantage of being minimally invasive and more convenient as DBSs do not require immediate sample processing (139). The use of DBSs is, additionally, an opportunity for cost reduction as no medically trained personnel are required (142). DBSs allow biological sample collection in nonclinical and field settings, including participants’ homes. DBS MMA may be an ideal screening tool for B-12 inadequacy in newborns as well as populations in remote settings.  22  Vitamin B-12 biomarker concentrations and related influencing factors during pregnancy Pregnancy-related physiological changes include hemodilution, hormonal changes, altered renal function, increased cell synthesis, and active and passive transport of nutrients across the placenta (10,118). Hemodilution refers to a disproportionate increase in plasma volume, including during pregnancy. It can lead to a decrease in red blood cell and biomarker concentrations in pregnant women (118).  The vitamin B-12 requirement has been suggested to increase during pregnancy due to rapid cell replication resulting from enlargement of the uterus, placental development, and fetal growth (10). Serum and urinary concentrations of the soluble form of the holoTC-receptor, CD320, with high abundance on the placenta, increase as pregnancy progresses (143). A significant increase of serum soluble CD320 concentrations from wk 15 to wk 35 of gestation has been reported among Danish pregnant women (n= 27) (143). This suggests enhanced B-12 transport across the placenta occurs throughout pregnancy. Further, intestinal absorption of B-12 has been suggested to be enhanced in pregnant women (98). Rodent studies suggested that placental lactogen may upregulate the expression of cubam receptors in the intestine (144). Cubam receptors are the binding-site of the intrinsic factor/B-12 complex in the intestine, which is the rate-limiting step in B-12 absorption (145). Such physiological changes, i.e., increased transport across the placenta and increased absorption, may affect circulating biomarker concentrations throughout pregnancy. Total B-12 concentrations decrease during pregnancy as shown in a variety of populations (Figure 1-2 and Table 1-4) (35,87,146–149). The decrease from the 1st to the 3rd 23  trimester appears to be consistent among the studies; the discrepancy in the pattern may partially be attributed to different methods used in assessing gestational age (150). The decrease in total B-12 concentrations throughout pregnancy could, in part, be explained by hemodilution (149). It has also been hypothesized that the drop in total B-12 concentration during pregnancy may be caused by a shift in B-12 binding proteins. A significant decrease in holohaptocorrin concentrations (87) has been reported while holoTC concentrations remained unchanged in 3 studies of pregnant women (total n= 197) (87,148,151). The function of holohaptocorrin in pregnancy is to date largely unknown (146,151). It has further been suggested that this drop in total B-12 concentrations may not truly reflect B-12 deficiency during pregnancy because concentrations of the functional biomarker MMA (35,87,146,152) remained largely unchanged throughout pregnancy. Only marginal increases of median MMA concentrations from the 2nd to the 3rd trimester have been reported in 2 independent studies of healthy Danish women [n= 406, 110 nmol/L to 113 nmol/L (146) and n= 27, 100 nmol/L to 160 nmol/L (87), respectively]. Murphy et al. (2007) found no significant difference in mean (10th–90th percentile) plasma MMA concentrations between pre-conception [120 (90–170) nmol/L] and at 8 gestational wk [110 (90–170) nmol/L] or 20 gestational wk [110 (80–150) nmol/L] in 92 pregnant Spanish women (149). Additionally, the percent change in mean plasma MMA concentration after correcting for hemodilution was not significant in these women. Plasma MMA concentrations were, however, significantly increased at 32 wk of gestation [140 (90–200) nmol/L], also after correcting for hemodilution. Yet, limitations of MMA as functional indicator of B-12 status in pregnant women have been discussed previously in this chapter (124,153). Two case-control studies (total n: 82 cases and 57 controls) reported no significantly elevated MMA or tHcy concentrations in pregnant women 24  (gestational age: ~28 wk) with total B-12 concentrations indicative of B-12 deficiency in non-pregnant adults (<148 pmol/L) (123,153). Further, women classified as B-12 deficient did not have lower hemoglobin concentrations, indicative of anemia, a functional outcome of B-12 deficiency in 1 of the studies (153). These findings imply that the decrease in total B-12 concentration across trimesters is not indicative of intracellular or functional B-12 deficiency. Taken together, non-pregnant cutoffs for total B-12 are not appropriate to be used in pregnant women and likely overestimate the prevalence of deficiency because of the natural decrease in total B-12 concentrations during healthy pregnancy.  Figure 1-2: Median or mean total vitamin B-12 (B-12) concentrations during pregnancy as reported in select studies since 2000, with fitted trend lines and traditional cutoffs (dotted horizontal lines) for B-12 deficiency (<148 pmol/L), suboptimal B-12 status (148-221 pmol/L), and B-12 adequacy (>221 pmol/L). The results are presented without between-subject variability given the heterogeneity in data presentation in the respective publications. Details on the studies are presented in Table 1-4. 25  Table 1-4: Characteristics of studies from Figure 1-2.1  Location Population n at trimester2 B-12 supplement use (%)2 P-trend3 Ref.    1st 2nd 3rd 1st 2nd 3rd   Koebnick (2002) Germany healthy 31 39 38 39 31 32 <0.0001 (148) Milman (2006) Denmark healthy 406 295 256 0 0 0 <0.0001 (146) Muthayya (2006) India healthy, diverse SES 136 154 152 0 0 0 - (54) Murphy (2007) Spain healthy, some smoker 88 90 90 0 1.1 28 <0.05 (149) Greibe (2011) Denmark healthy 27 27 27 0 0 0 <0.0001 (87) Hure (2012) Australia healthy  138 138  66 66 <0.01 (147) Duggan (2014) India diverse SES, some women with anemia  119 102  0 0 <0.05 (35) 1 Ref: reference; SES: socio-economic status; -: data not available. 2 Numbers below (1, 2, 3) indicate trimester. 3 Significance for decrease in total B-12 concentrations over time.  It stands out, that, compared to others, Duggan et al. (2014), Muthayya et al. (2006), and Hure et al. (2012) reported substantially lower total B-12 concentrations at any trimester during pregnancy (Figure 1-2). The studies by Duggan et al. (2014) and Muthayya et al. (2006) were located in India (Table 1-4). B-12 deficiency, related to diet and other factors, in populations living in India and other South Asian countries are commonly reported and will be discussed later in this chapter. Duggan et al. (2014) reported a low median (quartile 1; quartile 3) dietary intake of 1.40 µg/d (0.84; 2.11) B-12 [Estimated Average Requirement (EAR): 2.2 µg/d] during the 1st trimester (35). Correlation between dietary intake and B-12 was not reported. However, daily supplementation with 50 µg B-12 significantly increased total B-12 concentrations in the population. Thus, low perinatal B-12 status appears prevalent in Indian populations and may be related to the low dietary B-12 intake.  26  While MMA concentrations were reported to remain largely unchanged during pregnancy, tHcy concentrations have been found to describe a U-shaped curve throughout pregnancy (154,155). In a cohort of 522 Spanish pregnant women, plasma tHcy concentrations decreased from early pregnancy (<12 wk gestation) to 15 wk gestation, after which they significantly increased (154). Significantly increasing tHcy concentrations from mid- to late pregnancy have been described in several cohorts (148,156,157). Plasma tHcy concentrations are mostly determined by folate status in pregnant women (16,146). In the Danish cohort of 406 pregnant women, tHcy concentrations were only weakly correlated with total B-12 concentrations at 18 wk gestation (146), while being strongly predicted by folate status at 18 wk, 32 wk, and 39 wk gestation (157). As such, plasma tHcy concentrations provide only limited utility as biomarker of B-12 status in pregnant women. In summary, pregnancy leads to hemodilution and an increase in B-12 requirements which is potentially met by increased B-12 absorption. The pregnancy-related physiological changes can lead to altered B-12 biomarker concentrations. Total B-12 concentrations have been shown to decrease during pregnancy, which could, in part, be explained by hemodilution. This decrease, however, may not truly reflect B-12 deficiency. HoloTC and MMA concentrations remain largely unchanged, especially during early pregnancy, but may be affected by hemodilution. Thus, the use of non-pregnant cutoffs or reference values may not result in an accurate B-12 status assessment of pregnant women. Vitamin B-12 biomarker concentrations during infancy Adequate B-12 homeostasis at birth is understood to be comprised of metabolic B-12 sufficiency in combination with sufficient B-12 stores acquired throughout pregnancy (10). 27  The requirement of venous blood samples to assess B-12 biomarkers limits the possibilities to determine B-12 status in newborns. Venous blood draws in newborns are only recommended in clinical settings and for diagnostic purposes. Research studies commonly assess biomarkers in cord blood (158) as indicators of infant nutrient status to avoid newborn venous blood draws and lower the burden on study participants.  Mean cord blood concentrations of total B-12, MMA and tHcy have been reported to be significantly higher (up to 2 times) than maternal serum biomarker concentrations at birth (149,156,159). Additionally, in a study of 51 newborns, cord blood total B-12 concentrations were found to be 1.3 times the neonatal serum total B-12 concentrations (160). Accumulation of B-12 in the placenta or enhanced transport of B-12 across the placenta may be a reason for these findings (160) and suggest a preferential unidirectional transport of B-12 across the placenta. As such, cord blood total B-12 concentrations may overestimate neonatal B-12 status. Additionally, the unique composition of cord blood with a high concentration of stem cells, challenges in reliable sample collection, and the tight connection to the maternal metabolism may affect biomarker concentrations in cord blood samples. DBS samples collected between 24–48 hours after birth are much more reflective of neonatal metabolism (161). Infants have lower total B-12 and higher MMA concentrations than adults (116,162,163). A trend of changes in B-12 biomarker concentrations, indicative of a decreasing B-12 status from birth to 6 mo postpartum and subsequent increase has been reported in infants (116,162,163). A comprehensive Norwegian study described significantly increasing serum total B-12 and decreasing MMA and tHcy concentrations in apparently healthy infants (n= 118) from 6 to 12 mo of age (11% to 33% multivitamin use; some exclusively breastfed) (116). 28  Thereafter, total B-12 concentrations increased to a maximum at ~3–7 y after which they decreased to adult concentrations. MMA and tHcy concentrations remained consistent after age 1 y. Hay et al. (2008) likewise found significantly lower serum total B-12 and holoTC concentrations and significantly higher serum MMA and tHcy concentrations in infants (non-supplemented with B-12) aged 6 mo (n= 262) than infants aged 12 mo (n= 244), independent of breastfeeding status (163).  Taken together, infantile B-12 status appears to increase after 6 mo postpartum, the age at which complementary foods are often introduced (164). However, it is to date not understood if this observed increase in B-12 status is determined by the introduction of complementary foods, gut maturation, changes in renal function, or other unknown reasons.   Assessment of B-12 status in newborns or infants can be challenging due to the need for biological samples, age-specific cutoffs, and physiological changes affecting biomarker concentrations during infancy. Serum or urinary MMA concentrations, with or without additional measures of total B-12 concentrations, have been described as preferred indicator of infantile B-12 deficiency in clinical settings (68,110). Quantification of MMA concentrations compared, versus the assessment of only total B-12 concentrations, has the advantage of providing additional information on potential B-12 deficiency due to inborn errors of metabolism, as discussed above. Additionally, MMA has been described as a sensitive biomarker of B-12 status in infants (21,62,165). Specker et al. (1988) found that urinary MMA concentrations of exclusively breastfed infants of vegetarian mothers (n= 17), of whom 57% were B-12 deficient (total B-12 <148 pmol/L), were significantly higher (2.6–790.9 µmol/L creatinine) than of controls (n= 6; 1.7–21.4 µmol/L creatinine) (165). Similar findings have been demonstrated for infant plasma MMA concentrations (21). Honzik et al. (2010) reported 29  that only 1 of 40 infants who displayed clinical symptoms of B-12 deficiency and whose mothers were diagnosed with B-12 deficiency either due to malabsorption or low dietary intake had urinary MMA concentrations within the control range (<15 mg/g creatinine) (62). The authors, however, also commented that the specificity of urinary MMA was low as B-12 deficiency could not be confirmed by low plasma total B-12 concentrations (<148 pmol/L) in 10 of 16 infants with elevated urinary MMA concentrations (166). On the other hand, others have argued that MMA may be a specific marker of B-12 status in infants because impaired renal function and inborn errors of metabolism, both rare conditions in infants, are the only causes of markedly elevated MMA concentrations aside from B-12 deficiency (110,167). In summary, MMA is a sensitive indicator of B-12 status in infants and may provide some advantage over total B-12 in clinical settings. However, given the changes in B-12 biomarker concentrations, including MMA, during infancy, age-specific cutoffs are needed to assess infantile B-12 status. Lastly, it should be mentioned that some genetic polymorphisms, aside from inborn errors of B-12 metabolism, have been identified that alter B-12 biomarker concentrations (8). Yet, no single genotype has consistently been identified to have a substantial impact on B-12 status (134). The MTHFR C677T variant is the most commonly studied genotype in relation to B-vitamin metabolism. The homozygous TT compared with the CC genotype has been related to a greater risk of B-12 deficiency (total B-12 <148 pmol/L) in a study of healthy adults [n= 6,784; odds ratio (OR): 1.78; 95% CI: 1.25; 2.54] (168). However, no associations between the MTHFR C677T genotype and total B-12 or MMA concentrations have been observed in healthy South Asian (n= 55) and European (n= 151) women aged 19–35 y living in Vancouver (169) or in Canadian newborns (n= 368) (156). Additionally, the G allele of the 30  FUT2 A893G genotype has been associated with lower total B-12 concentrations (169). Variants of the transcobalamin gene (TCN2 C677G and A67G) can have been associated with low circulating holoTC concentrations (170,171) and altered tHcy (172) and MMA (173) concentrations, although not consistently (174). Recently, genome-wide sequencing of 2,210 healthy Irish adults revealed that 12% of the variability in circulating MMA concentrations is accounted for by mutations in HIBCH, the gene encoding for 3-hydroxyisobutyryl-CoA hydrolase which is part of valine metabolism, and in the gene encoding for acetyl-CoA synthase (ACSF3) (124). However, this study has yet to be repeated in other populations. In summary, certain genotypes may influence concentrations of some B-12 biomarkers, however, no single strong genetic predictor of B-12 status has been described.  Biomarker cutoffs and reference values4 for pregnant and pediatric populations Given the physiological differences between pregnant and pediatric populations with healthy adults, appropriate B-12 biomarkers and related cutoffs must be derived for accurate assessment of perinatal, neonatal and infantile B-12 status. The 2 most commonly reported reference values for all populations defining B-12 deficiency, also used by the Institute of Medicine (82) and the World Health Organization (WHO) (175), are total B-12 <148 pmol/L and <150 pmol/L (176) (Table 1-5). This reference value was first reported by Lindenbaum et al. (1994) who derived it from “the manufacturer’s stated lower limit of normal for the radioassay” and applied it to determine the prevalence of B-12 deficiency in older adults aged 65–99 y (177). It was later confirmed using inflection                                                  4 Reference values are values obtained from healthy populations against which measurements of patients or populations can be interpreted (327). Cutoffs on the other hand are well established reference values or reference values which have been evaluated against clinical measures of adverse health outcomes (12). 31  point analysis against MMA, as a functional outcome, in the NHANES 1988–1994 data (n= 33,994) (178). To date, the reference value has been well established in adult populations (114). Given that total B-12 <148 pmol/L is a well recognized reference value for B-12 deficiency in non-pregnant adults, it will be from here forth referred to as a cutoff. Table 1-5: Select cutoffs and reference values for adult, pregnant and pediatric populations for serum total vitamin B-12 (B-12), methylmalonic acid (MMA), and holotranscobalamin (holoTC) to determine vitamin B-12 deficiency (numbers in brackets indicate references). Bio- marker Adults Pregnancy (38,39,149,179) Infants (162,163) (86,114,180) 1st trimester  2nd trimester 6 months 12 months Total  B-12 <148 pmol/L <144 pmol/L1  <221 pmol/L3 <123 pmol/L1 <96 pmol/L2 <184 pmol/L3 <121 pmol/L2 <195 pmol/L4  <259 pmol/L2 <165 pmol/L2 <221 pmol/L4 <259 pmol/L2 HoloTC <35 pmol/L  <31 pmol/L1 <55 pmol/L2 <34 pmol/L1 <12 pmol/L5 <39 pmol/L4 <19 pmol/L5 <39 pmol/L4 MMA >370 nmol/L >210 nmol/L >170 nmol/L6  >150 nmol/L6 >290 nmol/L7 >2200 nmol/L7 >500 nmol/L8 >830 nmol/L7 >400 nmol/L8 1 Reference value determined as 10th percentile in apparently healthy, pregnant women.  2 Reference value determined as 2.5th percentile in apparently healthy pregnant women or infants.  3 Cutoff determined as statistically significant concentration to reduce neural tube defect-affected pregnancies.  4 Reference value determined as 5th percentile in apparently healthy, non-breastfed infants.  5 Reference value determined as 5th percentile in apparently healthy, breastfed infants.  6 Reference value determined as 90th percentile in apparently healthy, pregnant women.  7 Reference value determined as 95th percentile in apparently healthy, pregnant women or breastfed infants.  8 Reference value determined as 95th percentile in apparently healthy, non-breastfed infants.  Population-specific reference values for direct biomarkers of B-12 status have been computed from reference intervals of healthy, pregnant women (Table 1-5). A study of un-supplemented pregnant women (n= 92), residing in Spain, reported reference values (10th percentile), indicating B-12 deficiency, of plasma total B-12 <144 pmol/L and plasma holoTC <31 pmol/L at 8 wk of gestation and <123 pmol/L and <34 pmol/L at 20 wk of gestation, 32  respectively (149). In comparison, Milman et al. (2007) calculated a substantially lower reference value of plasma total B-12 <96 pmol/L at 18 wk of gestation as 2.5th percentile in a population of medium to high socio-economic status, un-supplemented, pregnant Danish women (n= 441) who delivered healthy infants (birth weight >2500 g) at ≥37 wk of gestation (179). This data suggests that in pregnant women total B-12 concentrations above lower reference values than in adult populations may indicate B-12 adequacy, which stands in agreement with the finding that total B-12 concentrations decrease during healthy pregnancy. Despite this, in epidemiological studies in pregnant women, serum total B-12 concentration of <148 pmol/L (the same value as for non-pregnant adults) is commonly used to determine the prevalence of B-12 deficiency (156,181,182).  On the other hand, adverse health outcomes related to perinatal B-12 inadequacy have been described at maternal total B-12 concentrations above these reference values. Data of 3 independent case-control studies of Irish women (n: 278 NTD cases; 901 controls) allowed Molloy et al. (2009) to extrapolate a cutoff of serum total B-12 <221 pmol/L at 28 d (4 wk) of gestation to prevent NTD-affected pregnancies (39). A similar study conducted in Ontario, Canada, (n: 317 NTD cases; 434 controls) found an increased risk for NTD-affected pregnancies [OR (95% CI): 2.0 (1.1; 3.9)] when women had serum holoTC concentrations ≤55.3 pmol/L [lowest quartile; compared to highest quartile (>121.0 pmol/L)] at 15–20 wk of gestation (38). The neural tube closes at 28 d of gestation; a time point when many women are yet not aware that they are pregnant. Thus, to prevent NTD-affected pregnancies, these studies suggest that women should have circulating total B-12 or holoTC concentrations above the cutoff at conception or before pregnancy. 33  Values for MMA defining B-12 deficiency range from >210 to >480 nmol/L (114,176). MMA concentrations >370 nmol/L in non-pregnant adults has been described as a “generally agreed on cutoff for elevated plasma MMA” (84). The value was derived from the reference value corresponding to the 97.5th percentile of healthy adults aged 40–68 y (n= 58; mean ± SE serum total B-12: 277±21 pmol/L ) (183). The reference value (95th percentile) of B-12 replete (serum total B-12 >50th population percentile) participants in NHANES 1999–2000 (non-pregnant individuals aged ≥3 y) with normal renal function (serum creatinine ≤133 µmol/L for men and ≤115 µmol/L for women) was substantially lower at 210 nmol/L (84). Given the much larger sample size and more diverse population in NHANES, MMA concentrations >210 nmol/L may provide a more accurate reference value for healthy, non-pregnant individuals aged ≥3 y. However, it should be noted that the MMA reference value of >210 nmol/L represents the 95th percentile of the population, not the 97.5th as has been recommended for clinical purposes (184), and the reference population may be biased as only individuals with the highest B-12 status were included.  In pregnant women, substantially lower reference values of >150 nmol/L during the 1st trimester and >170 nmol/L during the 2nd trimester have been suggested, determined as the 90th percentile of apparently healthy, pregnant women, residing in Spain (n= 92) (149). The lower values may, in part, be explained by hemodilution, although, hemodilution during the 1st trimester has been described to be no more than 10% (118).  Two studies computed age-specific reference intervals in pediatric populations, dependent (163) or independent (162) of breastfeeding status. Recently, data from a comprehensive trial (CALIPER) of 257 ethnically diverse, partially supplemented and breastfed infants (aged 5 d to 1 y) residing in Canada yielded a reference value according to 34  clinical standard procedures (2.5th percentile, CLSI C28-A3) of serum total B-12 <191 pmol/L (95% CI: 134; 202 pmol/L) for B-12 deficiency (162). MMA concentrations decrease substantially during infancy, especially in breastfed infants (116,163). No reliable reference values or cutoffs indicative of B-12 deficiency are available for MMA in infants. To summarize this section (1.2.2.), 4 B-12 biomarkers with limited specificity and sensitivity are available. No consensus has yet been reached on the most valid method of B-12 status assessment. HoloTC is the form of B-12 which is available to tissues, including the placenta. It has been suggested to be a more sensitive indicator of B-12 status. However, no study has yet determined the sensitivity of holoTC in pregnant women. Plasma tHcy concentrations have only limited utility as a B-12 biomarker because tHcy concentrations are primarily determined by folate, rather than B-12, status. MMA is the most specific functional indicator of B-12 status. Given the minimally invasive collection, DBS MMA may be a convenient tool for B-12 status assessment in vulnerable populations, including newborns.   Biomarker concentrations during pregnancy and infancy can be affected by physiological changes, including hemodilution and maturation, respectively. A decrease in concentrations of most biomarker of B-12 status during pregnancy has been shown. Total B-12 concentrations were found to decrease substantially. Additionally, MMA and tHcy concentrations were found to decrease substantially from 6 mo to 12 mo of life in infants, while total B-12 concentrations increased. The predictors of these changes are not fully understood. Pregnancy- and age-specific reference values, and eventually well established cutoffs, are required for accurate B-12 status assessment in pregnant women and infants, respectively. Several studies have suggested reference values obtained from reference populations. Yet, more research is needed to evaluate these reference values.  35  1.2.3. Predictors and Prevalence of Vitamin B-12 Deficiency During Pregnancy and Infancy in Canada and World-Wide Predictors of low vitamin B-12 status  Vitamin B-12 is found in animal-source foods, fortified foods and supplements only. Increased prevalence of B-12 deficiency has been reported among many populations with low intake of animal-source foods (185–187). Long-term (>3 y) lacto-ovo vegetarians have been reported to not meet their Estimated Average Requirement (2.2 µg/d) for B-12 during pregnancy (187). In the same study, mean (25th–75th percentile) serum total B-12 concentrations were significantly lower (P< 0.05) in lacto-ovo vegetarians (n= 60) than omnivores (n= 108) during the 1st [176 (100–317) pmol/L and 249 (201–319) pmol/L], 2nd [176 (102–271) pmol/L and 238 (190–305) pmol/L], and 3rd trimesters [127 (90; 184) pmol/L and 169 (141; 213) pmol/L]. Similar trends were reported for holoTC and tHcy. Diets with a limited diversity of animal-source foods have been associated with lower B-12 status in pregnant women. Cultural, ethical, religious, and economical reasons have been described for low intake of animal-source foods (6,188). This and other reasons, e.g. gastro-intestinal diseases leading to malabsorption, render some ethnic minorities, for example South Asians, and populations in low- and middle-income countries (LMIC) at increased risk for B-12 deficiency. South Asians are the largest ethnic minority in Canada and the second largest minority in Vancouver (48). Cultural, economic, and geographic restraints may limit access to diverse foods and adequate prenatal education during pregnancy among this minority (189). Prevalence of B-12 deficiency in non-pregnant South Asian populations in Canada and the 36  United States was the subject of a limited number of studies (190–192). A lower B-12 status of South Asians compared to healthy, omnivorous Europeans has, however, consistently been reported in Canada (191,192). In a study in Metro Vancouver, we found a higher prevalence  of inadequate B-12 status (serum total B-12 <220 pmol/L) among reproductive-aged (19–35y) women of South Asian (40%; n= 55) compared to women of European ethnicity (32%; n= 149), yet the difference was not statistically significant (P= 0.17) (169). However, the study did not have sufficient power to detect a statistically significant difference due to the low number of women of South Asian ethnicity. Additionally, in a recent cross-sectional study of 320 pregnant women (20–35 wk of gestation) in Metro Vancouver, we revealed that the odds for being B-12 deficient (total B-12 <148 pmol/L; 18% of the study population) were 10-times higher among women who self-identified as South Asian (8% of the study population) (193). Given the high overall prevalence of inadequate B-12 status (34%) in women of reproductive age residing in Metro Vancouver (169), Vancouver women and those of South Asian ethnicity may be at high risk for perinatal B-12 deficiency. In comparison, the prevalence of  inadequate B-12 status (total B-12 <220 pmol/L) among non-pregnant, Canadian women aged 20–45 y (n= 1,576; Canadian Health Measures Survey 2007–2009) was 25.3% (194). Prenatal B-12 containing supplement use is prevalent in Canada and an important contributor to perinatal B-12 intake (156,181,195). In a study in pregnant women (n= 368) in Ontario, Canada, prenatal supplement use was 60.1% (95% CI: 55.8; 64.3) before conception, which increased to 92.8% (95% CI: 89.6; 95.2) during early pregnancy (≤16 wk of gestation) (195). Women received a daily dose of 2.6 µg (IQR: 2.6; 12.0) B-12 before conception (n= 191) and 2.6 µg (2.6; 10.0) B-12 during early pregnancy (n= 312) from supplements alone (RDA: 2.6 µg/d). Being born in Canada [OR: 2.16 (95% CI: 1.34; 3.49); P= 0.002] and higher 37  education [OR: 1.93 (95% CI: 1.05; 3.57); P= 0.03] were positively associated with increased pre-pregnancy supplement use. Immigrant women living in Canada have been reported to experience discrimination, class inequities and social isolation (196), which may be prohibitive for the access to prenatal supplements and health care. Thus, the potentially lower use of prenatal supplements may render immigrant women in Canada at an increased risk for B-12 inadequacy. Populations in LMIC and resource-poor settings have been found to suffer from the double burden of low intake of animal-source foods as well as high rates of infections (5,6). Low intake of animal-source food may be due to lack of availability, high cost, and cultural or religious beliefs. Diets low in animal-source foods are associated with a higher prevalence of micronutrient deficiencies, including B-12, iron, and zinc (197). B-12 deficiency in these populations is of particular concern, because B-12 is only found in animal-source foods, fortified foods, and supplements (198). However, some may also be found in tempe (199), a fermented soy-product, typically eaten in Indonesia. Additionally, infections or gastrointestinal morbidity may be caused by poor food hygiene and food preparation practices and lead to malabsorption of nutrients (200). Helicobacter pylori is a common food-borne pathogen in certain populations in LMIC (201,202). Infection with helicobacter pylori has been associated with lower B-12 status (203). This, other infections, and low intake of animal-source foods may in part explain the high prevalence of B-12 deficiency reported in some LMIC (5).  Indonesia is a LMIC in South-East Asia. In Indonesia, no data on prevalence of B-12 deficiency or B-12 intake are available. Dietary diversity among Indonesian children under 5 y was characterized as medium to low (204). Among infants of 6–11 mo, 47.7 % (95% CI: 44.04; 51.31) did not receive foods from 4 or more food groups per day (minimum dietary 38  diversity), 62.3 % (95% CI: 58.78; 65.73) were not fed a minimum of 4 or more times per day (minimum food frequency), and 35.4 % (95% CI: 31.73; 39.14) did not receive minimum dietary diversity and minimum food frequency (205). Additionally, breastfed infants between 6 and 23 mo from poor, rural households were significantly less likely to receive adequate complementary feeding [OR= 1.76 (95% CI: 1.16; 2.68)] compared to urban infants (206). Overall, dietary surveys among infants in Indonesia indicate poor feeding practices. Additionally, intake of animal-source and nutrient-dense foods are low, especially among the rural population (207–209), and rates of infection are high (210). Thus, many predictors of B-12 deficiency have been described in Indonesian infants rendering them at risk for B-12 deficiency. Predictors of neonatal and infantile vitamin B-12 deficiency It is hypothesized that maternal perinatal B-12 deficiency is a predictor of offspring B-12 deficiency (159). The etiology of acquired infant and child B-12 deficiency, however, is not yet fully understood. It has been discussed that newborns of B-12 deficient mothers have developed insufficient B-12 stores in utero, that can be depleted within 1 year postpartum if untreated (211). Breastmilk of B-12 inadequate mothers provides insufficient B-12 to restore infant B-12 status (35,212–214). This suggests a strong association between maternal perinatal B-12 status and neonatal and infant B-12 status, respectively. Longitudinal (152,156,215) and cross-sectional (159,216–218) studies have described the relationship between circulating total B-12, holoTC, MMA, and tHcy concentrations in the mother during pregnancy and at delivery, respectively, and B-12 biomarker concentrations in her newborn. Cord blood has been assessed as proxy for neonatal B-12 status in these studies. 39  Its limitations have been discussed above (Chapter 1.2.2). Murphy et al. (2007) conducted the most comprehensive longitudinal trial assessing multiple biomarkers at multiple time points (149). Significant correlations between maternal plasma MMA concentrations at 8, 20, and 32 gestational wk, with cord blood MMA concentration at birth [ρ (P): 0.29 (<0.05), 0.28 (<0.05), and 0.40 (<0.01), respectively] have been described in this trial of mostly un-supplemented women (n= 92) in Spain (149). Maternal plasma total B-12 concentrations at 8, 20, and 32 gestational wk were not correlated with cord blood MMA concentration. Only maternal plasma holoTC concentration at 32 wk of gestation, but not at 8 or 20 wk of gestation, was correlated with cord blood MMA concentration (ρ= -0.51; P< 0.0001). This suggests that MMA throughout all pregnancy and holoTC during late pregnancy may be more sensitive indicators of functional B-12 status of the offspring at birth. In a Canadian cohort of mostly supplemented women (n= 368), maternal serum total B-12 (ρ= 0.71; P< 0.0001) and plasma tHcy (ρ= 0.66; P< 0.0001), but not plasma MMA (ρ= 0.26; P= 0.65), concentrations assessed at early (0–16 wk of gestation) and mid- to late (23–37 wk of gestation) pregnancy were predictors of the corresponding cord blood biomarker concentrations (156). The discrepancy between the studies may in part be explained by the different sample size, statistical approach (simple correlation analysis vs multivariable regression analysis) or supplement intake. Thus, it appears there is a significant association between maternal B-12 status during pregnancy and neonatal B-12 status assessed at birth. However, it is not yet understood which B-12 biomarker assessed during pregnancy best predicts neonatal B-12 status. Maternal B-12 status, breastfeeding duration and frequency, and dietary B-12 intake from complementary foods have been described as predictors of infant B-12 status. Maternal and infant circulating total B-12 concentrations were correlated [standardized β: 0.27; P< 40  0.0001 and r= 0.31; P< 0.05, respectively] at 12 mo postpartum in 2 studies in Guatemala (total n: 270 and 183, respectively) (219,220). Additionally, infant B-12 status (serum or plasma total B-12 concentration) was correlated with B-12 intakes from complementary foods [standardized β: 0.30; P< 0.0001 and r= 0.22; P< 0.05, respectively]. Thus, infants receiving complementary foods poor in B-12 or being breastfed by B-12 deficient mothers may be at risk for B-12 deficiency. Breastmilk, especially of women in LMIC with high prevalence of B-12 deficiency, has been identified as an insufficient dietary B-12 source for infants (35,163,212–214,220). In Kenya, infants aged 1–6 mo (n= 286) were estimated to receive 0.12 µg/d B-12 from breastmilk (AI: 0.4 µg/d) (212). Frequency of breastfeeding has been negatively associated (standardized estimate: -0.29; P< 0.0001) with infant B-12 status (n= 304) at 12 mo in a resource-poor setting in Guatemala where 49 % of infants and 68 % of mothers were B-12 inadequate (plasma total B-12 <220 pmol/L) (219). In a Norwegian prospective trial, healthy, breastfed infants (n= 224) had significantly lower serum holoTC concentrations compared with infants receiving infant formula at 6-mo of age [37 (95% CI: 33; 40) and 74 (95% CI: 64; 86) pmol/L] and 12-mo of age [51 (95% CI: 46; 56) and 76 (95% CI: 70; 80) pmol/L] (163). Exclusive breastfeeding is recommended for the first 6 mo postpartum (164), making infants of B-12 deficient mothers most vulnerable for B-12 deficiency.  Prevalence of perinatal vitamin B-12 deficiency in Canada Reports of B-12 deficiency among pregnant Canadian women range from non-existent (221) to 43 % (156), depending on biomarker and gestational age. Nationally representative data for non-pregnant women are available from the Canadian Health Measures Survey 41  (CHMS; Cycle 1.1 2007–2009) (194). Results from the Canadian Health Measures Survey and other Canadian trials are summarized in Table 1-6. Nearly 5% of all Canadians were described to be B-12 deficient (serum total B-12 <148 pmol/L) and 18.5% had suboptimal B-12 status (serum total B-12: 148–220 pmol/L). Women aged 20–45 y, have a 25% prevalence of inadequate B-12 status (serum total B-12 <221pmol/L). The CHMS does not include pregnant women; thus, there are no nationally representative data on B-12 status in pregnant women. In Ontario, a comprehensive, cross-sectional study of women aged 15–46 y (n= 10,622), found 6.9% of non-pregnant and 5.2% of pregnant women (gestational age <4 wk) to be deficient in B-12 (serum total B-12 <125 pmol/L) (222). In the same study, prevalence of deficiency increased to 10% in pregnant women at >28 wk of gestational age.  42   Table 1-6: Vitamin B-12 status in Canadian reproductive-aged and pregnant women.1 Location Study subjects n B-12 deficiency Inadequate B-12 status Ref.    Prevalence Indicator2 Prevalence Indicator2  Canada3 Non-pregnant women aged 20–45 y  1,576 5.0%  total B-12 <148 pmol/L 25.3%  total B-12 <220 pmol/L (194)  Vancouver 16 wk of gestation  264 10%  total B-12 <148 pmol/L 21%  total B-12 <220 pmol/L (181)  36 wk of gestation  264 23% total B-12 <148 pmol/L 35%  total B-12 <220 pmol/L  Alberta first trimester  123 0.8% (n= 1) holoTC3 <35 pmol/L -  (221)   second trimester  521 1.2% (n= 6) holoTC3 <35 pmol/L -   Toronto 12-16 wk of gestation  340 17%  total B-12 <148 pmol/L 38%  total B-12 <220 pmol/L (156)  delivery (28-42 wk)  292 35%  total B-12 <148 pmol/L 43%  total B-12 <220 pmol/L  Ontario <28 d of gestation  1,244 5.2%  total B-12 <125 pmol/L -  (222)  >28 d of gestation  2,490 10%  total B-12 <125 pmol/L -   Newfoundland ~16 wk of gestation  1,424 25% total B-12 <130 pmol/L -  (182) 1 Ref: reference; y: years; B-12: vitamin B-12; wk: weeks; holoTC: holotranscobalamin; -: data not available. 2 Indicator used to determine B-12 status. Prevalence in pregnant women may be interpreted with caution as estimated using cutoffs (total B-12) and reference values (holoTC) for non-pregnant adults. 3 Canadian Health Measures Survey Cycle 1.1 (2007-2009). 3 holoTC quantified in EDTA-plasma, results should be interpreted with caution.43  Circulating total B-12 concentrations have been shown to naturally decrease during healthy pregnancy (147–149), as described above. Thus, the use of non-pregnant adult cutoffs of serum total B-12 to determine B-12 deficiency in pregnant women may overestimate the prevalence of deficiency. The use of alternate biomarkers becomes important. Visentin et al. (2015; Toronto; ) determined plasma MMA concentrations in addition to serum total B-12 as indicator of perinatal B-12 status (156). During early pregnancy (12–16 wk of gestation), 17% of women had serum total B-12 concentrations <148 pmol/L (B-12 deficient), while only 1.9% had elevated plasma MMA concentrations (>271 nmol/L). Prevalence of deficiency increased to 38% and 5.3% of women, respectively, at delivery (35–37 wk of gestation). Using MMA as biomarker of B-12 status resulted in a substantially lower prevalence of deficiency in this study of pregnant women in Toronto, Canada. As discussed above, serum total B-12 concentrations <148 nmol/L may be overestimating the prevalence of deficiency. Given that MMA concentrations appear to remain largely unchanged during pregnancy, MMA concentrations, especially during early pregnancy when the effect of hemodilution is smaller, may be a better estimate to determine B-12 deficiency. Using this biomarker, B-12 deficiency appears to be low in this cohort of highly educated and high socioeconomic status Canadian women. Fayyaz et al (2014) and the APrON (Alberta Pregnancy Outcomes and Nutrition) study team reported the lowest prevalence of B-12 deficiency in pregnant women in Canada. Using plasma holoTC concentration <35 pmol/L as reference value, the prevalence of B-12 deficiency in pregnant women of high socioeconomic status was 0.8% (1/123) and 1.2% (6/521) during the 1st and 2nd trimesters, respectively. However, the study should be interpreted with caution. HoloTC was quantified in EDTA plasma which was shown to positively bias holoTC measurements by 40% (106) and likely underestimated the prevalence of B-12 44  deficiency. Further, holoTC concentrations above 35 pmol/L have been associated with adverse health outcomes in pregnant women and non-pregnant adults (38,95). Thus, the B-12 deficiency in the APrON participants might have been underestimated.  In conclusion, only limited studies are available focusing on B-12 deficiency in pregnant women in Canada. Studies assessing multiple biomarkers and using pregnancy-specific reference values are necessary to draw any conclusions and support interventions. Additionally, no study has yet focused on vulnerable population groups, such as South Asians, the largest ethnic minority in Canada, who may be at increased risk for perinatal B-12 deficiency. 1.3.Summary of Rationale Vitamin B-12 is required for cell synthesis and neurocognitive development, 2 physiologic processes that are most active during rapid stages of growth (31,32). Prolonged inadequate B-12 status during pregnancy and infancy have been associated with a wide array of adverse health outcomes in the mother and her offspring, including long-term lower cognitive scores (7). Thus, identifying and treating pregnant women, newborns, and infants with B-12 deficiency is crucial for maternal health, fetal and infantile development, and long-term cognitive health. The availability of multiple and reliable biomarkers and related reference values or cutoffs is crucial to facilitate reliable assessment and sensitive screening for perinatal, neonatal, and infantile B-12 deficiency (130). Today, 4 B-12 biomarkers with limited sensitivity and specificity are available. Total B-12 is the most commonly used direct biomarker of B-12 status (114). HoloTC, which reflects the B-12 readily available for uptake by tissues including the 45  placenta, has been suggested to be a more sensitive indicator of B-12 status (95). Elevated circulating MMA concentrations indicate intra-cellular B-12 deficiency in adults and infants (21,22,187). MMA is regarded as the most specific indicator of functional B-12 status because tHcy (anoth possible functional biomarker) is influenced by folate and other B-vitamins (114). No consensus has yet been reached on valid assessment of B-12 status. Methylmalonic acid can be quantified in DBSs (122,135); however, methods are not sensitive enough to detect subtly elevated MMA concentrations as seen in mild inborn errors of metabolism or nutritional B-12 deficiency (122). DBSs are a convenient and minimally invasive tool for collection of biological samples (139,141). The use of DBSs allows for sample collection in newborns and remote populations (141). DBS MMA may have the potential to allow for screening of B-12 deficiency in vulnerable populations. Some studies have shown that holoTC and total B-12 perform equally well in diagnosing adults with B-12 deficiency (99,101). Others have suggested that holoTC has a better diagnostic capacity than total B-12 (104). The performance of either biomarker to diagnose pregnant women with B-12 deficiency has not yet been explored. Physiological changes during pregnancy affect perinatal biomarker concentrations (10,147). It has been suggested that a decrease in total B-12 concentrations during pregnancy does not truly reflect B-12 deficiency (87). Thus, pregnancy-specific reference values, and eventually cutoffs, need to be established to allow for accurate measurement of B-12 status in pregnant women. Low intakes of animal-source foods have been discussed as potential predictors of B-12 deficiency (6,219). Population groups living in LMIC, including Indonesia, and certain ethnic groups, especially South Asians, are vulnerable to B-12 deficiency (6,191,192,206). 46  While an overall high prevalence of micronutrient deficiencies has been described in Indonesian infants (223), nothing is known about their B-12 status. Additionally, we know only little about pregnant South Asian women living in Canada. A tendency to a higher prevalence of B-12 deficiency in reproductive aged women of South Asian ethnicity residing in Metro Vancouver has recently been described (169). South Asians are Canada’s largest ethnic minority (48) and may be at increased risk for B-12 deficiency during pregnancy. Establishing and using reliable tools to screen for B-12 inadequacy during pregnancy and infancy can identify populations or individuals at risk for perinatal and infantile B-12 deficiency. This will allow for future targeted interventions and may eventually reduce the prevalence of B-12 inadequacy and its related adverse health outcomes during rapid stages of growth. 1.4.Research Objectives and Hypotheses With the overall goal to facilitate and apply screening for perinatal, neonatal, and infantile B-12 inadequacy, the objectives of this thesis are as follows: 1. (A) to develop a sensitive assay for quantitative analysis of MMA in DBSs, (B) to test its applicability by comparing DBS MMA with plasma MMA concentrations, the current reference indicator, in a convenience sample of healthy adults, and (C) to assess MMA stability in DBSs at various storage temperatures to provide recommendations for sample handling in the field; 2. (A) to calculate a reference interval of DBS MMA in healthy, term newborns and (B) to test the specificity of the reference interval by analyzing its ability to identify cases with and without inborn errors of B-12 metabolism; 47  3. (A) to compare the serum total B-12 concentrations between pregnant women of South Asian and European ethnicity residing in BC, (B) to determine the diagnostic performance of holoTC compared to total B-12 during pregnancy in detecting functionally B-12 deficient (as indicated by elevated MMA concentrations) pregnant women, and (C) to compute reference values for total B-12 and holoTC, indicative of B-12 deficiency in pregnant women.  4. (A) to determine the prevalence of B-12 deficiency in infants aged 6–12 mo living in rural Indonesia, and (B) to identify predictors of B-12 status in these infants.  The objectives have the following hypotheses: 1. A. Null Hypothesis: The LC-MS/MS assay for quantitation of MMA is not sensitive enough [limit of detection (LOD) > lowest concentration] to detect MMA concentrations found in extracts of DBSs of healthy adults. A. Research Hypothesis: MMA can be quantified in DBSs of healthy adults using LC-MS/MS with a limit of detection of >3 signal-to-noise ratio and baseline-resolution. B. Null Hypothesis: DBS MMA and plasma MMA concentrations of healthy, non-pregnant young adult women are not correlated. B. Research Hypothesis: DBS MMA and plasma MMA concentrations of healthy, non-pregnant young adult women correlate significantly (P< 0.05) and the nature of the correlation is linear. 48  C. Null Hypothesis: DBS MMA concentrations remain unchanged over time when stored at -80°C, refrigerator temperature (2–4°C), room temperature (~20°C), or 35°C for up to 1 y. C. Research Hypothesis: DBS MMA concentrations change significantly (P< 0.05) over time when stored at -80°C, refrigerator temperature (2–4°C), room temperature (~20°C), or 35°C for up to 1 y. 2. A. Null Hypothesis: It is not feasible to quantify MMA in routinely collected DBSs of healthy, term newborns. A. Research Hypothesis: MMA quantified in routinely collected DBSs of healthy, term newborns is distributed in a manner that allows calculating a reference interval according to current clinical standards [CLSI C28-A3 (184)]. B. Null Hypothesis: DBS MMA concentrations of all cases – with or without inborn error of B-12 metabolism – lie within the limits of the reference interval. B. Research Hypothesis: DBS MMA concentrations of cases with inborn errors of B-12 metabolism are higher than the upper limit of the reference interval; DBS MMA concentrations of cases without inborn errors of B-12 metabolism lie within the upper and lower limits of the reference interval. 3. A. Null Hypothesis: There is no difference in B-12 status (serum total B-12 concentration) between women of South Asian and European ethnicity living in Metro Vancouver, during the 1st and 2nd trimesters, and their newborns (DBS MMA concentration).  A. Research Hypothesis: Pregnant women of South Asian ethnicity  have a significantly lower B-12 status than pregnant women of European ethnicity, as indicated by serum total B-12 concentration assessed during the 1st and 2nd trimesters; newborns born to women of 49  South Asian ethnicity have a significantly lower functional B-12 status than newborns born to women of European ethnicity, as indicated by DBS MMA concentrations assessed at birth.  B. Null Hypothesis: Maternal serum total B-12 and serum holoTC concentrations, assessed during the 1st and 2nd trimesters of pregnancy, respectively, perform equally in detecting women (i) who have elevated MMA concentrations (> 210 nmol/L) or (ii) who have newborns with elevated circulating MMA concentrations (DBS MMA > reference interval determined in objective 2A).  B. Research Hypothesis: Maternal holoTC concentrations, assessed during the 1st and 2nd trimester of pregnancy, respectively, perform significantly better than maternal total B-12 concentrations in detecting women (i) who have elevated MMA concentrations (> 210 nmol/L) or (ii) who have newborns with elevated circulating MMA concentrations (DBS MMA > 75th percentile). C. Null Hypothesis: There is a non-significant or linear relationship between maternal serum MMA and serum total B-12 or holoTC concentrations during the 1st and 2nd trimester. C. Research Hypothesis: The relationship between maternal serum MMA and serum total B-12 or holoTC concentration during the 1st and 2nd trimester can be described by a linear-splines model allowing for the calculation of an inflection point. 4. A. Null Hypothesis: The prevalence of infants aged 6–12 mo living in rural Indonesia who have plasma total B-12 concentrations <191 pmol/L (162) is low (<5%). A. Research Hypothesis: The prevalence of infants aged 6–12 mo living in rural Indonesia who have plasma total B-12 concentrations <191 pmol/L is above 5%. 50  B. Null Hypothesis: Plasma total B-12 and MMA concentrations of these infants are not predicted by infant age nor by maternal DBS MMA concentrations. B. Research Hypothesis: Plasma total B-12 and MMA concentrations of these rural Indonesian infants are significantly predicted by infant age and/or by maternal DBS MMA concentrations.  51  2. Methylmalonic Acid Quantified in Dried Blood Spots Provides a Precise, Valid, and Stable Measure of Functional Vitamin B-12 Status in Healthy Women 2.1.Summary Methylmalonic acid (MMA) is a sensitive and specific functional biomarker of vitamin B-12 (B-12) status, commonly assessed in plasma or serum. Dried blood spots (DBSs) allow simpler and more cost-efficient blood sampling than plasma. To facilitate convenient testing for vitamin B-12 deficiency in large-scale surveys and in population groups from remote areas, I developed a method for MMA quantification in DBSs and tested its applicability as well as the long-term stability of MMA in DBSs at various temperatures. MMA was extracted from an 8-mm DBS punch with water:methanol (95:5, v:v) and methyl-d3-malonic acid as the internal standard. After sample cleanup by ultrafiltration and hexane extraction, MMA was quantified by using reversed-phase LC-tandem mass spectrometry. Extraction conditions were optimized to maximize the detection signal and achieve DBS extract concentrations above the lowest limit of quantification (signal-to- noise ratio ≤10) of 10 nmol/L. Recovery was between 93% and 96%. Intra- and interassay variation (CV) for DBSMMA was 0.49% and 2.3%, respectively. Calibrators showed linearity (R2= 0.998) between 10 and 10,000 nmol/L. In 94 healthy women, MMA concentrations in DBS extract (min-max: 10.2–80.5 nmol/L) and plasma (min-max: 68–950 nmol/L) were correlated (r= 0.90) (P< 0.001). MMA concentrations in DBSs were stable at room temperature (18–22°C) for 1 wk, in the refrigerator for 8 wk, and at -80°C for at least 1 y. This simple and robust method allows quantification of MMA in DBSs of healthy individuals. The linear relation between plasma and DBS MMA suggests that DBS MMA could predict plasma MMA, the current reference indicator for functional B-12 deficiency. With the advantages of minimally invasive specimen collection and no need for 52  laborious blood processing steps, this method has the potential to be a reliable, convenient, and field-applicable alternative for assessment of B-12 status. 2.2.Introduction Low B-12 status has been associated with an increased risk of adverse pregnancy outcomes (34,36), poor cognitive performance in children (76), and accelerated cognitive decline in the older adults (102). The prevalence of inadequate B-12 status is common worldwide (224) and reported to be ~20% in the United States (84,130) and Canada (194) (serum total B-12 <220 pmol/L), 48% in Indian children (plasma total B-12 <200 pmol/L) (225), and up to 70% in Latin American countries (serum/plasma total B-12 <221 pmol/L) (226). In light of the potential health implications and the high prevalence of B-12 deficiency, the availability of a robust and convenient assessment tool is crucial for monitoring B-12 status in various population groups with the goal of preventing B-12 deficiency.  Methylmalonic acid is the most specific functional biomarker and a sensitive indicator for B-12 deficiency (85,114,125). B-12 is required as a cofactor for the interconversion of methylmalonyl-CoA to its isomer succinyl-CoA in odd-chain fatty acid metabolism. In case of B-12 deficiency, methylmalonyl-CoA is converted into MMA and released into circulation. An increase in circulatory MMA reflects intracellular B-12 deficiency. Plasma (or serum) is the most commonly used body fluid to determine MMA concentration (85,126).  Dried blood spot analysis was developed as a diagnostic tool for newborn screening in the 1960s (139,227). Its application was later suggested for collection of biologic data in large national surveys and for use in remote settings (139,228). For DBS analysis, blood from a finger prick, or a heel prick in newborn screening, is applied to a specific filter paper that 53  stabilizes analytes and proteins. This minimally invasive and simple method can be conducted in the field and by individuals in nonclinical settings resulting in the advantage of lower cost, lack of venipuncture, and no need for immediate sample processing and special storage (139).  Methylmalonic acid is a dicarboxylic acid quantified by isotope dilution technique by using gas chromatography (GC) or liquid chromatography (LC) coupled with single (MS) or tandem (MS/MS) mass spectrometry (112,113,122,135,229–233). Methods for MMA determination in DBSs have so far been developed in the context of newborn screening for the diagnosis of methylmalonic acidemia (122,135,229), an inborn error of metabolism that leads to 100–1000-fold higher blood MMA concentrations than healthy controls (229). The required and achieved limits of detection for these methods are insufficient for assessment of normal MMA concentrations and thus B-12 status in healthy populations.  Thus, the objectives of the study were as follows: (A) to develop a sensitive assay for quantitative analysis of MMA in DBSs, (B) to test its applicability by comparing DBS MMA with plasma MMA concentrations, the current reference indicator, in a convenience sample of healthy adults, and (C) to assess MMA stability in DBSs at various storage temperatures to provide recommendations for sample handling in the field. The overall goal was to develop a minimally invasive, reliable, and convenient tool for assessing B-12 status in large-scale surveys and remote field settings. 54  2.3.Materials and Methods 2.3.1. Reagents and Chemicals All chemicals met the requirements of the American Chemical Society (ACS-grade), unless otherwise stated. MMA was purchased from Sigma-Aldrich; deuterium-labeled methyl-d3-malonic acid (d3-MMA) from Cambridge Isotope Laboratories; succinic acid and formic acid from Acros Organics (Thermo Fisher Scientific); and hexanes from Fisher Scientific (Thermo Fisher Scientific). Methanol and water in LC-MS grade were purchased from Fluka (Sigma-Aldrich) for use as mobile phase. Purified water for standard and sample preparation was prepared by using Milli-Q equipment (EMD Millipore, Merck KGaA) and had a conductivity of <18 mΩ. 2.3.2. Preparation of Calibrators Calibrators for dried blood spot analyses Stock solutions of 100-mmol/L MMA, succinic acid, and d3-MMA were prepared separately in purified water and stored at -80°C until analysis. Calibration curves were produced with 9 points from 0–100 nmol/L by adding 100 mL of 80-nmol/L d3-MMAworking solution as the internal standard to 100 mL of each calibrator solution. Succinic acid concentrations were 100-times the concentrations of MMA to mimic a DBS sample. The standards were acidified with 20 mL of 4% formic acid before analysis.   55  Calibrators for plasma analyses Calibration curves were produced as described above but with 7 points from 0–500 nmol/L. Succinic acid was added in 10-times the concentration of MMA to mimic a plasma sample. The internal standard had a final concentration of 400 nmol d3-MMA/L. 2.3.3. Collection of Dried Blood Spot and Plasma Specimens Finger-prick DBS and venous whole blood samples were obtained at a fasting state from a convenience sample of 94 healthy women (aged 19–35 y) in metropolitan Vancouver, BC, Canada, who participated in a descriptive cross-sectional study (169). All subjects gave written informed consent prior to blood sampling. The Clinical Research Ethics Board of the University of British Columbia approved the study protocols.  After pricking either the index or middle finger with a safety lancet, the first blood drop was discarded to avoid contamination of the DBS sample with tissue cells. Five DBSs were then created on protein saver cards (Whatman 903; Thermo Fisher Scientific). For method development, validation, and stability testing, DBS aliquots were created by applying 50 μL EDTA-treated whole blood onto protein saver cards. There was no difference (i.e., value > intra-assay CV) in MMA concentration between extracts from DBSs created with finger-prick blood and those made with EDTA-treated whole blood. The DBSs were allowed to dry overnight at room temperature (~21°C) before being stored in sealable plastic bags with desiccant packs at -80°C (or alternate temperatures for stability testing, as explained below).  56  For testing of method applicability, plasma samples were obtained after centrifugation of subjects’ whole blood samples at 1400 x g for 15 min at 4°C and stored at -80°C until analysis. A plasma pool was created from 3 subjects and used for quality control. 2.3.4. Sample Processing for Methylmalonic Acid Extraction Sample processing for dried blood spot analysis MMA was extracted from an 8-mm DBS punch in 150 μL extraction solution consisting of equal volumes of 10% methanol and 80-nmol/L d3-MMA working solution by mixing in a vortex for 1.5 h. The extract was deproteinized by ultrafiltration by using Amicon Ultra Centrifugal Filters (EMD Millipore, Merck KGaA) with a mass cutoff of 3 kDa. Fifty μL of the filtrate was acidified with 5 μL 4% formic acid and underwent a liquid-liquid-extraction step with hexane (3:1) for removal of lipids and other nonpolar compounds. Ten μL of the cleaned sample extract was used for injection into the LC-MS/MS system. Sample processing for plasma analysis Plasma sample preparation was conducted by using a method which was developed by adapting the method of Blom et al. (112) to include more thorough sample cleanup procedures. Briefly, 100 μL of plasma was mixed with 100 μL of 800-nmol/L d3-MMA working solution and deproteinized by ultracentrifugation (112) by using a mass cutoff of 3 kDa. After acidification with 4% formic acid, the filtrate underwent a liquid-liquid-extraction step with hexane. Injection volume into the LC-MS/MS system was 10 μL of the cleaned sample extract. 57  2.3.5. Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry System Chromatographic separation of the isomers MMA and succinic acid was conducted on an Agilent 1260 LC instrument (Agilent Technologies) by using a reversed-phase C-18 column (2.1 x 50 mm, 3.5 µm, 120 Å; InertSustain; GL Sciences) at 40°C. A C-18 guard column (2.1 x 10 mm; Fortis) was attached to the analytic column. Elution was achieved with an isocratic flow of 0.4% formic acid in water:methanol (95:5, v:v) at a flow rate of 200 µL/min and followed by a wash step (Table 2-1).  Table 2-1: Liquid chromatography conditions and diagram of flow for quantification of methylmalonic acid in dried blood spots.1 Time (min) Flow (µL/min) Mobile Phase A: 0.4% formic acid in water (%) Mobile Phase B:  0.4% formic acid in methanol (%) Direction of flow Purpose 0.00 200 95 5 Waste Elution of salts 1.00 200 95 5 MS Elution of analyte 3.00 200 95 5 MS  3.10 400 5 95 Waste Wash step 5.00 400 5 95 Waste  5.10 400 95 5 Waste Conditioning 8.00 400 95 5 Waste  8.10 200 95 5 Waste  12.00 200 95 5 Waste  1MS: mass spectrometer.  The effluent was directed into an ABSciex API4000 triple-quadrupole mass spectrometer (AB SCIEX Pte.) used in selected reaction monitoring mode. The electrospray ionization probe was operated in negative mode; source and compound parameters are described in Table 2-2. Following the isotope dilution principle, MMA concentration was quantified by using the isotope ratio (analyte peak area/internal standard peak area). Analyst 58  software (version 1.5.2; AB SCIEX Pte.) was used for LC-MS/MS control, data acquisition, and data processing. Table 2-2: Source and compound parameters for API 4000 (AB SCIEX Pte.) for quantification of methylmalonic acid.1 Source Parameter Compound Parameter Collision gas (psi) 5 Declustering potential (J) -35 Curtain gas (psi) 30 Entrance potential (J) -10 Ion source gas 1 (psi) 50 Collision energy (J) -12 (MMA) Ion source gas 2 (psi) 50  -16 (d3-MMA) Ion spray voltage (V) -4500 Collision cell exit potential (J) -5 Temperature (°C) 600   1 MMA: Methylmalonic acid; d3-MMA: methyl-d3-malonic acid.  2.3.6. Method Validation Experiments Matrix effect Ion suppression or enhancement caused by the matrix (matrix effects) (234) was tested by comparing the analyte response (Area Under the Curve; AUC) in aqueous solution and after addition to a quality control sample. In brief, a post-extraction spike sample was created by adding 2 µL of 1 mmol/L aqueous MMA solution (2 nmol) to 100 µL DBS extract of a quality control sample [concentrations (c) ~28.6 nmol/L MMA] to achieve a final concentration of ~ 48.6 nmol/L. The mean AUC (n= 5) of an aqueous solution of 50 nmol/L MMA was compared to the mean AUC (n= 5) of the post-extraction spike sample. Additionally, the post-column infusion method was applied to test for a matrix effect. In brief, an aqueous solution of 50 nmol/L MMA was constantly infused into the MS/MS system (operated in single reaction monitoring mode). The change in signal was monitored when injecting a blank DBS extract.   59  Recovery All experiments were performed in triplicate following the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines on Validation of Analytical Procedures Q2(R1) (235). Low-, medium-, and high- quality controls were created by adding 1, 2, and 3 μL of 10-mmol/L MMA, respectively, to 50 μL whole blood before creating the DBSs. The approximation that 50 μL whole blood creates a DBS with an 11-mm diameter allowed estimating the recovery. Imprecision To determine the intra-assay CV, 5 replicates of the DBS and plasma quality control samples were prepared and injected 5 times. The inter-assay CV was determined by analyzing 5 DBS and 5 plasma replicates (each injected 5 times) on 3 consecutive days. In addition, duplicate preparations of DBSs and plasma quality control samples were included in each analytic run to test for long-term variability. Limits of detection and quantification The limit of detection and lowest limit of quantification were determined by injection of serially diluted standards. The limit of detection was defined as the lowest MMA standard concentration with a signal-to-noise ratio ≥3 in 5 consecutive injections. The lowest limit of quantification was defined as the lowest MMA standard concentration with a signal-to-noise ratio ≥10 (235). 60  2.3.7. Testing for Long-term Stability of Methylmalonic Acid Concentrations in Dried Blood Spots Batches of DBS aliquots from 10 subjects were stored at the following temperatures and locations: 37°C (incubator); 18–23°C (temperature- controlled room); 2–4°C (refrigerator); and -80°C (laboratory-grade deep freezer). DBS MMA was measured in duplicate analysis at baseline (i.e., 24 h after sample collection) and after 1, 2, 4, 6, 8, 12, 15, 19, 23, 36, and 51 wk of storage, depending on the storage temperature and respective impact on stability. 2.3.8. Statistical Analyses Values in the text for method validation data are presented as means ± SDs. For the applicability testing, MMA concentrations of DBS extract and plasma from 94 women were tested for normality by using the Shapiro-Wilk test (236). Because of positive skewness, MMA concentrations are presented as medians and IQRs. The relation between DBS extract and plasma MMA concentrations was tested by calculating the Spearman rank correlation coefficient (ρ). Simple linear regression analysis (r) was performed to create a linear equation between DBS extract and plasma MMA for potential predictive purpose. Stability of MMA in DBSs was defined as no significant change from baseline. The Student’s paired t test was used to compare the differences in MMA concentrations of DBS extract to baseline. Results were considered significant at P< 0.05. All analyses were conducted by using SigmaPlot for Windows software (version 11.2; Systat Software). 61  2.4.Results 2.4.1. Method Development and Optimization The analyte extraction conditions were optimized to maximize the extraction yield and the detection signal. The analyte extraction resulted in a higher yield and signal intensity of MMA with a buffer containing methanol and water (5:95, v:v) in the composition of the mobile phase than with water only (Figure 2-1). The extraction yield saturated after 1.5 h with no further increase in MMA yield. The volume of extraction solution was minimized to a total of 150 μL without impairing the precision of the assay.   Figure 2-1: Yield of mean (SD) dried blood spot methylmalonic acid (DBS MMA) concentration (n= 10) after varying extraction time and using either extraction buffer [methanol:water (v/v): 5:95] or water. 62  Chromatographic separation of the isomers MMA and succinic acid in DBS extract was achieved as shown in a representative chromatogram in Figure 2-2. Despite the high concentrations of succinic acid in DBS extract, the presented method separates MMA from succinic acid by using a 50-mm C-18 reversed-phase column and 0.4% formic acid in water:methanol (95:5, v:v) as the mobile phase. The addition of 0.4% formic acid to the mobile phase led to the highest signal intensity of MMA compared with using other concentrations of formic acid (0.1% or 4%) or the addition of acetic acid to the mobile phase. The best ionization and signal intensity of MMA was achieved by using a negative ionization mode. 63   Figure 2-2: Chromatogram of a dried blood spot extract containing [A]: Succinic acid (SA) and methylmalonic acid (MMA), mass to charge ratio (m/z) of 117→73; and [B]: Internal standard, methyl-d3-malonic acid (d3-MMA), m/z of 120→76. 64  An analyte peak width at 50% intensity of 0.14 ± 0.003 min for MMA from DBS extract and 0.12 ± 0.004 min for plasma MMA was achieved with isocratic elution. The use of a gradient flow did not improve chromatographic separations independent of the flow-rate, time sequence of gradients, and mobile phase composition. LC-column life time was substantially increased by thorough sample cleanup including deproteinization (3 kDA cutoff) and extraction of nonpolar compounds as well as by the application of a guard column. Under these conditions, the column has shown a life time of at least 1500 analyses. The integration of an LC-column wash step directed into waste after analyte and internal standard elution prevented potential ion suppression and contamination of the ion source. 2.4.2 Method Validation Matrix effect The mean (95% CI) AUC of the aqueous standard (c= 50 nmol/L MMA; n= 5) of 131,400 (121,758; 141,042) units was not different from the mean (95% CI) AUC of the post-extraction spiked sample (c~ 48.6 nmol/L MMA) of 123,800 (96,733; 150,867) units. Additionally, no decrease in signal was observed when performing the post-column infusion experiment (data not shown). As such, standards were prepared in aqueous solutions. Limit of detection, lower limit of quantification, and linearity The limit of detection for MMA was determined to be 1.25 nmol/L (0.148 ppb). No signal above the limit of detection was detected at the retention time of MMA when extracting clean Whatman 903 filter paper. The lower limit of quantification was determined to be 65  10 nmol/L (1.180 ppb). The method had a high degree of linearity (R2 = 0.998) over at least 3 orders of magnitude (10–10,000 nmol MMA/L) (Figure 2-3).  Figure 2-3: Linear range for aqueous methylmalonic acid (MMA) standards measured by liquid chromatography-tandem mass spectrometry. Figure in box depicts an enlargement of standards at low concentrations. Recovery and imprecision The recovery for DBS MMA was 93–98% (Table 2-4). Intra-assay CVs for DBS MMA were <0.5% on all 3 days (Table 2-4). The between-run imprecision tested on 3 consecutive days was 2.3%; the inter-assay variability over 1 y was 6.3%. With respect to between-sample preparation variation, the CVs of duplicate analyses were mostly <1% and none exceeded 3%. 66  Table 2-3: Recovery for the quantitative analysis of methylmalonic acid (MMA) in dried blood spot (DBS) extract.1  MMA concentration in extract (150 μL) (nmol/L) MMA concentration in whole blood2  (nmol/L) MMA added3 (μmol) Recovery (%) DBS 44.1±5.0 250±51 - - Low recovery 68.8±2.0 368±9.3 10 95.4-97.3 Medium recovery 112±6.1 632±28 20 92.9-93.8 High recovery 152±3.0 856±14 30 95.1-98.0 1 Values are mean ± SD. 2 Calculated using the estimation that 50 μL form a DBS with 11 mm in diameter. 3 Added to 50 μL of whole blood. Table 2-4: Imprecision for the quantitative analysis of methylmalonic acid (MMA) in dried blood spot (DBS) extract and plasma.1  Intra-assay variation (%) Inter-assay variation (%) MMA concentration (nmol/L) MMA in DBS extract       DBS quality control I 0.49 2.3 28.6±0.22    Low recovery 0.66 - 68.8±2.0    Medium recovery 1.9 - 112±6.1    High recovery 1.4 - 152±3.0    DBS control II (long-term)3 - 6.3 34.9±1.3 MMA in plasma       Plasma quality control I 0.79 1.2 196±4.4    Plasma quality control II                       (long-term)3 - 3.0 192±6.1 1 Values are ranges, CVs, or mean ± SD. 2 Mean ± SD represent data from inter-assay variation analysis. 3 Long-term variability testing was conducted for 1 year.  2.4.3 Method Applicability DBS extract and plasma MMA concentrations were correlated (ρ= 0.90; P< 0.001) in 94 healthy women. Figure 2-4 illustrates the scatterplot with DBS extract MMA as the predictor of plasma MMA concentrations (r= 0.97; P< 0.001). After excluding subjects with 67  elevated plasma MMA concentrations [defined as >271 nmol/L (130)], the data of 84 subjects were normally distributed and had a linear relation between plasma and DBS extract MMA concentrations (r= 0.84; P< 0.001).   Figure 2-4: Scatterplot of plasma methylmalonic acid (MMA) concentrations vs. dried blood spot (DBS) extract MMA concentrations of healthy women (n= 94) indicating a linear relation [(r= 0.97), P< 0.001]. Horizontal line at y= 271 nmol/L represents reference value distinguishing between elevated and normal MMA concentrations.  The median (IQR) MMA concentration in DBS extracts of 94 women was 17.7 nmol/L (14.6; 22.4). With the approximation that 50 μL whole blood forms a DBS 11 mm in diameter, the median (IQR) MMA concentration in whole blood was estimated to be 200 nmol/L (165; 68  252). The median (IQR) plasma MMA concentration was 158 nmol/L (119; 216) in these women. 2.4.4 Long-term Stability of Methylmalonic Acid in Dried Blood Spots Figure 2-5 shows the mean ± SD difference from baseline in MMA concentration of DBS extract (n= 10), as measured over the time span of 1 y of specimen storage at different temperatures. DBS MMA concentration increased significantly (P< 0.05) when stored at 37°C right after baseline, at room temperature after 1 wk, and at 2–4°C after 8 wk of storage. When stored at -80°C, DBS MMA remained stable for (at least) 1 y.  Figure 2-5: Differences from baseline in the methylmalonic acid (MMA) concentration of dried blood spot (DBS) extract after storage of DBS at various temperatures and locations for up to 1 year.  Values are means ± SDs, n= 10. Different from baseline: *P< 0.05, **P< 0.01, ***P< 0.001; Student’s paired t-test. c: concentration.  69  2.5 Discussion This study presents a simple, robust, and validated method for MMA quantification in DBSs by using LC-MS/MS. The assay achieved sufficient sensitivity to quantify DBS MMA in subjects with plasma MMA concentrations in the normal range. To my knowledge, this is the first method to quantify DBS MMA of healthy individuals. The applicability of the method was demonstrated in healthy women showing a linear relation between plasma and DBS MMA concentrations.  Currently available assays for MMA determination in DBSs have been developed for newborn screening as a 2nd-tier test in the diagnosis of methylmalonic acidemia (122,135,229). These methods are designed for measurement of MMA at 100–1000-fold higher concentrations than those determined in healthy controls. The method by la Marca et al. (135) was shown to be capable of monitoring MMA and diagnosing methylmalonic acidemia in newborn DBSs. However, because the assay’s lower limit of quantification was above MMA concentrations of unaffected neonates, this method does not have the sensitivity to be used for quantitative analysis of MMA with the goal of assessing vitamin B-12 status. Turgeon et al. (122) developed a method for MMA quantification by using LC-MS/MS after derivatization of MMA to butylesters. The limit of detection for MMA was 130 nmol/L, which also is above the required lower limit of quantification for MMA quantification from DBSs of healthy individuals. The presented method achieves a lower limit of quantification of 10 nmol/L and thus had sufficient sensitivity to quantify MMA in DBSs of healthy women. In addition to adult populations, the presented method is ideally suited for application to newborn screening of at-risk populations (e.g., offspring of vegetarian mothers and South Asians). Moreover, it 70  could represent an ideal 2nd-tier test for confirmation of methylmalonic acidemia, reducing the number of false-positive results of routinely applied methods. Determination of low-mass molecules in highly diluted samples requires instrument sensitivity and highly consistent instrument performance. Column durability and duration of MS/MS sensitivity was maximized by employing thorough sample cleanup procedures, by using a precolumn, and including a wash step eluting into waste. The wash step prevented possible fluctuations in sensitivity from slowly eluting substances, which can occur with consecutive sample injections and no wash step as reported by Blom et al. (112). The presented method achieved a consistent MS/MS sensitivity over 3000 injections. The column wash step and reconditioning, however, increased the analytic run time. Injection-to-injection time could be shortened by performing the column wash offline. The method applicability study showed that the relationship between DBS and plasma MMA concentrations can be described by a simple linear regression, allowing for an easy conversion from DBS to plasma MMA. However, I acknowledge that some regression assumptions were violated, as the residuals were not normally distributed. Plasma MMA was positively skewed, as reported in representative population-based studies (111).In these healthy women, plasma MMA concentrations ranged between 68 and 950 nmol/L, reflecting vitamin B-12 adequacy [defined as a plasma MMA concentration between 60 and 271 nmol/L (84,130)] and presumed functional B-12 deficiency [defined as a plasma MMA concentration >271 nmol/L (130)]. In clinical settings and the majority of population-based studies, B-12 status is most commonly assessed by using serum total B-12 for reasons of assay convenience and availability. The sensitivity of this direct indicator, however, has been argued (126). To address its limitations, an expert panel suggested the use of both a direct and functional 71  biomarker of B-12 status, ideally serum total B-12 and MMA (126). However, uncertainties exist about the appropriate cut-offs for both indicators, which may contribute to potential misclassification of individuals as shown by Bailey et al. (130). Repletion trials in different age groups would be the ideal study design to evaluate plasma and DBS MMA reference values that accurately reflect B-12 status.  The ideal storage temperature for MMA in DBSs was shown to be -80°C after storage for at least 1 y. At all higher temperatures, DBS MMA increased over time, most rapidly at 37°C. Storage of DBSs at inappropriate temperatures can thereby lead to falsely elevated MMA concentrations that would result in potential misclassification of subjects as B-12 deficient. The presented data suggest that DBSs for MMA analysis can be stored (in a sealed bag with a desiccant pack) at room temperature (18–22°C) for 1 wk and in the refrigerator for 8 wk. These results are consistent with reports that methylmalonyl- CoA mutase, the enzyme catalyzing the interconversion of MMA and succinic acid, is still active at 5°C (237) and stays active after being stored at -20°C for 6 mo (238). Long-term stability of MMA concentration in DBSs stored at -80°C is maintained, allowing for retrospective analysis of deep-frozen DBS specimen (e.g., from newborn screening). Although MMA is a sensitive indicator and the most specific functional biomarker for B-12 deficiency, circulatory MMA is affected by renal function. Nevertheless, the inclusion of MMA as a biochemical indicator is strongly suggested for vitamin B-12 status assessment in national surveys, such as the NHANES (126). The application of DBSs could considerably simplify collection of biologic data for large-scale surveys and enable specimen collection in rural or developing region studies. To control for renal function and thereby enhance specificity of MMA as a vitamin B-12 biomarker, renal function could additionally be 72  monitored in dried specimen. McCann et al. (239), for example, described a method for the determination of creatinine in dried urine samples. 2.6 Conclusions In conclusion, a simple and sensitive method that allows quantification of MMA in DBSs of healthy individuals is presented. The method was validated by verifying its linearity and by achieving a sufficient lower limit of quantification as well as high precision and accuracy. The method had excellent performance and robustness. DBS MMA concentrations remained stable for 8 wk when stored at 2–4 °C and for at least 1 y when stored at -80 °C. Research is needed for the derivation of reference ranges and cutoffs for DBS MMA in various population groups. Given the linear relation of DBS and plasma MMA, DBS MMA concentrations could be used to predict plasma MMA concentrations and thereby evaluate B-12 status. Because it avoids venipuncture, blood processing steps, and immediate storage requirements, this method has the potential to be a robust, convenient, and field-applicable alternative for B-12 status assessment. 73  3. Reference Interval of Methylmalonic Acid Concentrations in Dried Blood Spots of Healthy, Term Newborns to Facilitate Neonatal Screening of Vitamin B-12 Deficiency 3.1.Summary Unrecognized vitamin B-12 (B-12) deficiency in early infancy can lead to poor development of the child. Methylmalonic acid (MMA) is the most specific functional biomarker of B-12 status. To facilitate timely diagnosis and treatment of neonatal B-12 deficiency, the objective of this study was to calculate a reference interval of MMA in dried blood spots (DBSs) of healthy, term newborns. MMA was quantified in 160 newborn DBSs, routinely collected for newborn screening, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The reference interval of DBS MMA was calculated according to current guidelines (CLSI EP28-A3c). The effect of storage at room temperature on DBS MMA concentrations was determined. The mean (95% CI) DBS MMA concentration of 160 healthy, term newborns was 16.8 (15.9-17.6) pmol/8-mm spot. The reference interval (2.5th to 97.5th percentile) for MMA was 9.89 to 29.3 pmol/8-mm spot (0.450 to 1.33 µmol/L whole blood). DBS MMA concentrations increased over time when stored at room temperature (p< 0.005). DBS MMA concentrations of children with methylmalonic acidemia (n= 4) were above the upper limit of the reference interval, while DBS MMA concentration of 1 child with propionic acidemia was within the reference interval, demonstrating specificity of the interval. This is the first study to present a reference interval for DBS MMA of healthy, term newborns utilizing a new highly sensitive MMA method.  Analysis of DBS MMA collected during newborn screening may have the potential to identify newborns with acquired B-12 deficiency. Newborn DBS MMA concentrations increase with storage at room temperature, therefore, sample storage has to be monitored for evaluation of DBS MMA data. 74  3.2.Introduction Vitamin B-12 (B-12) plays an essential role in DNA synthesis, methylation of biomolecules, and odd-chain fatty acid metabolism, which are important processes during growth and brain development (32). Prolonged B-12 deficiency during infancy has been associated with negative long-term health outcomes in children (34), including impaired cognitive function and developmental delay (71,78). Infantile B-12 deficiency often presents with nonspecific symptoms, such as hypotonia, irritability, or failure to thrive (32,70). Thus, diagnostic testing may be delayed until more obvious signs of B-12 deficiency develop (70). Timely diagnosis and treatment of infantile B-12 deficiency is crucial to prevent permanent neurological injury. In Canada, newborns and young infants are not routinely assessed for B-12 status unless they display obvious clinical features of severe deficiency. Maternal B-12 deficiency during pregnancy is a strong predictor of neonatal B-12 deficiency (35,147,159). Inadequate maternal dietary intake and malabsorption are the most common causes of infantile B-12 deficiency. Dietary patterns with reduced intake of animal sourced foods, such as vegetarian, vegan, or low-cost diets, and religiously or culturally motivated avoidance of animal products, are associated with an increased risk for B-12 deficiency (6,152,187). Additionally, gastrointestinal disorders, gastric bypass and pernicious anemia can lead to malabsorption and deficiency of B-12 (6,66). An Ontario study reported that 10% of pregnant women (> 28 days) were classified as B-12 deficient (serum B-12< 125 pmol/L) (222). Increased rates of up to 51% of pregnant women classified as B-12 deficient (total B-12 <150 pmol/L) have been found for India (35), Australia (147) and Mexico (240), potentially leading to a high prevalence of neonatal B-12 deficiency. Unrecognized neonatal B-12 deficiency worsens if the infant is exclusively breastfed by a B-12 deficient 75  mother due to decreased fetal stores and insufficient intake through breastmilk (70,214). Thus, assessment of neonatal B-12 status is crucial in populations with increased risk for maternal B-12 deficiency. Vitamin B-12 is required in odd-chain fatty acid and branched chain amino acid metabolism for the isomerization of methylmalonyl-CoA to succinyl-CoA. Intracellular B-12 deficiency leads to an increase in methylmalonyl-CoA which is converted into methylmalonic acid (MMA) and is released into the blood stream. Plasma MMA concentration thus serves as a functional biomarker of B-12 status and reflects intracellular B-12 deficiency (114). Compared to serum total B-12, MMA is also a sensitive indicator of B-12 deficiency: circulating MMA concentrations are increased in cases of acquired B-12 deficiency due to dietary restrictions or malabsorption (22,241). In individuals with inherited disorders involving MMA metabolism, blood MMA ranges from only mildly to markedly elevated (2 to 1000-fold). MMA is quantified in dried blood spots (DBS) during second-tier testing in a few newborn screening programs to improve test performance for inherited disorders, collectively known as methylmalonic acidemias (67,122,135). Yet, these published methods lack sufficient sensitivity to identify newborns with subtler increases in MMA concentrations and therefore might miss many cases of B-12 deficiency and some inherited disorders involving MMA metabolism. The reports of low frequency of neonatal B-12 deficiency detected through newborn screening, with and without second-tier testing, suggest an under-ascertainment (66,67,147,242). A highly sensitive method to quantify MMA in DBSs of healthy individuals using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been described in Chapter 2. This method may have the potential to screen newborns for acquired B-12 deficiency in a single analysis.  76  Diagnosis and treatment of neonatal B-12 deficiency at birth is crucial for healthy infant development. The importance of including infantile B-12 status in newborn screening panels has recently been emphasized in the literature (64). With the overall goal to facilitate screening and testing of neonatal B-12 status, the aim of this study was to calculate a reference interval of DBS MMA in healthy, term newborns. This will allow for the identification of newborns with subtly elevated DBS MMA concentrations and has the potential to identify newborns with acquired B-12 deficiency using routinely collected neonatal DBSs. 3.3.Materials and Methods 3.3.1. Study Samples Newborn DBSs are routinely collected in birthing hospitals across British Columbia and transported to the newborn screening laboratory at BC Children’s Hospital (Vancouver BC, Canada).  DBSs (n=160) for this study were randomly selected between December 2014 and April 2015 following routine newborn screening. DBSs were de-identified and stored at   -80°C for 2 mo, in accordance with the storage and use policy of the newborn screening program. Time from receipt of newborn DBSs at the laboratory until storage at -80 °C was noted and is referred to as time at room temperature. To reduce bias from prolonged transit time, only DBSs collected within birthing hospitals in the Greater Vancouver area were included in the study. Newborns with a birth weight <2500 g, diagnosed with any inherited disorders, receiving parenteral nutrition or transfusion as well as multiple births were excluded. Additionally, residual DBSs (n= 5) from children diagnosed with a variety of inherited disorders involving MMA metabolism were retrieved. DBSs of these patients are routinely 77  collected for monitoring purposes, and residual spots were de-identified and retrieved for MMA quantification. Storage of these patient DBS samples was at room temperature. The Clinical Research Ethics Board of The University of British Columbia (Vancouver BC, Canada) and the Newborn Screening Research Review Committee at BC Children’s Hospital (Vancouver BC, Canada) approved the study protocols. A waiver of consent was given for the release of the de-identified residual DBSs. 3.3.2. Biochemical Analyses Methylmalonic acid was quantified in DBSs using stable isotope dilution LC-MS/MS as described in Chapter 2.3. In brief, MMA was extracted from an 8-mm DBS punch. After sample clean-up, chromatographic separation was performed on an Agilent 1260 High Performance Liquid Chromatography instrument (Agilent Technologies) using a reversed-phase C-18 column (InertSustain, GL Sciences). MMA was quantified on an API4000 triple-quadrupole mass spectrometer (AB SCIEX Pte.).  3.3.3. Statistical Analysis After quantification, results were obtained as MMA (μmol) per L extract and converted into MMA (pmol) per 8-mm DBS punch. To calculate the concentration of MMA in whole blood, I assumed that an 8-mm DBS punch contains 22 μL of whole blood after testing the distribution of whole blood on the filter paper (data not shown). Normality of data was tested using Shapiro-Wilk test (236). Effect of newborn DBS MMA concentration on birth weight was determined using multivariate regression with sex as 78  confounding factor. Birth weight between male and female newborns was compared using two-sample t-test. All reference intervals were calculated according to the CLSI guideline EP28-A3c (184). The reference interval for all newborn data (n= 160) was computed as 2.5th and 97.5th percentile (parametric method) after Box-Cox transformation. Separate reference intervals for newborn DBS MMA stored for less and more than 7 days at room temperature, respectively, were calculated using the robust method (243,244) as advised for samples n <120 (184).  Effect of storage time at room temperature on newborn DBS MMA concentration was tested using Spearman’s rank correlation and visualized by a linear prediction plot. Mean DBS MMA stored for less (<168 h; n= 88) and more (>168 h; n= 72) than 7 days at room temperature was compared using Wilcoxon rank-sum test.  If not stated otherwise, results are presented as geometric mean with 95% confidence interval (95% CI) and tests were considered significant at P< 0.05. All statistical analyses were performed in Stata 14.1 (SataCorp) for Windows 8. Reference intervals were calculated in MedCalc 15.11 (MedCalc Software bvba). 3.4.Results 3.4.1. Sample Characteristics Dried blood spots of 160 newborns were randomly selected after completion of newborn screening. Mean (95% CI) birth weight was 3388.7 (3317.7; 3459.7) g and 43% (n= 69) of the newborns in this sample were male. Mean (95% CI) MMA concentration was 16.8 (15.9; 17.6) pmol in 8-mm DBS spots (Figure 3-1). Tukey’s test identified 5 outliers who 79  had no common characteristics, i.e., sex or birth weight. Mean (95% CI) MMA concentration in newborn DBS extracts was 0.112 (0.106; 0.117) µmol/L and ranged from 0.0357 µmol/L to 0.341 µmol/L. Thus, with a previously reported lower limit of quantification of 0.01 μmol/L for this method (Chapter 2.4.2), reliable quantification of MMA in all newborn DBS extracts was possible. Assuming 22 μL of whole blood from an 8-mm spot, mean (95% CI) MMA concentration in newborn whole blood was 0.764 (0.723; 0.800) µmol/L.  Figure 3-1: Tukey box plot of methylmalonic acid in 8-mm neonatal dried blood spots (pmol) with upper and lower limit of the reference interval (solid grey lines) and their 95% confidence interval (dashed grey lines) according to CLSI EP28-A3c (184).  There was no significant association between MMA and birth weight after controlling for sex. Sex was weakly but significantly associated with birth weight (r= 0.16; p< 0.05). Male newborns were a mean (95% CI) of 145 (3.24; 287) g heavier than females (p< 0.05). 80  3.4.2. Reference Interval Upper and lower limit of the reference interval are presented for MMA in the 8-mm DBS in pmol (Figure 3-1). Data of MMA in newborn DBSs were not normally distributed before (p< 0.001) and after removal of outliers by Horn’s algorithm (p< 0.05) (245); normal distribution was achieved through Box-Cox transformation according to CLSI 28-A3c (184). The mean (95% CI) upper limit (97.5th percentile) was 29.3 (27.0-32.0) pmol per 8-mm newborn DBS and the mean (95% CI) lower limit (2.5th percentile) was 9.89 (9.44-10.4) pmol per 8-mm newborn DBS. The reference interval for whole blood MMA concentrations was determined to have a mean (95% CI) lower limit of 0.450 (0.429; 0.473) μmol/L and a mean (95% CI) upper limit of 1.33 (1.23; 1.45) μmol/L. Four newborns (2.5%) displayed DBS MMA concentrations above the upper limit of the reference interval. Upper limits of reference intervals computed using alternative methods suggested by CLSI EP28-A3c – nonparametric percentiles method and normal method after normalizing the data by inverse transformation – did not differ significantly from the upper limit computed after Box-Cox transformation (Figure 3-2).  81   Figure 3-2: Comparison of different upper and lower limits of reference intervals, computed according to methods proposed by current guidelines CLSI EP28-A3c (184).  The primary reference interval was calculated as a central 95% interval (normal theory) after Box-Cox transformation to achieve normality of the data (primary recommendation). Alternate reference intervals were calculated using normal theory after inverse (1/x) transformation to achieve normality of the data (Shapiro-Wilk; p= 0.06) and the nonparametric percentiles method. The upper and lower limits of the reference intervals did not differ significantly. Error bars indicate 95% confidence interval.   3.4.3. Impact of Storage Time at Room Temperature on Dried Blood Spot Methylmalonic Acid Concentrations Newborn DBSs were stored at room temperature between 1 to 16 days (25.4 to 382 h), during the routine process of newborn screening, before being transferred to a -80 °C freezer. 82  MMA in newborn DBSs showed a small but significant correlation with the time DBSs were stored at room temperature (Spearman’s rho= 0.21; p< 0.01; Figure 3-3).   Figure 3-3: Correlation between methylmalonic acid in 8-mm dried blood spot (pmol) and time dried blood spot was stored at room temperature (h) and linear prediction with 95% confidence interval. Dashed, vertical line depicts 7 days (168 h).  Mean (95% CI) MMA in newborn DBSs, which had been stored at room temperature for less than 7 days (n= 88), was 15.8 (14.8; 16.8) pmol per 8-mm DBS and 0.718 μmol (0.673; 0.764) μmol/L whole blood. This was significantly lower than mean (95% CI) MMA in newborn DBSs, which had been stored at room temperature for more than 7 days [17.9 (16.6; 19.3) pmol per 8-mm DBS and 0.814 (0.755; 0.877) μmol/L whole blood; n= 72; P< 0.005]. However, upper limits did not differ significantly when separate reference intervals were computed for MMA in newborn DBSs stored for less and more than 7 days at room temperature, respectively (Figure 3-4). 83   Figure 3-4: Upper and lower limit of the reference intervals computed for methylmalonic acid in dried blood spots (DBSs) stored for <7 days (n= 72) or ≥7 days (n= 88) at room temperature (rt; 18–21°C) using the robust method. Error bars indicate 95% confidence interval.  3.4.4. Comparison with Control Samples Methylmalonic acid was quantified in 5 DBS samples of children who are being treated and monitored for inborn errors of metabolism. Sample characteristics are displayed in Table 3-1. DBS MMA concentrations were above the upper limit of the reference interval in all samples of patients with methylmalonyl-CoA-mutase deficiency or cobalamin C deficiency. As expected, the patient with propionyl-CoA carboxylase deficiency had MMA concentrations in the normal range (used as negative control).  In the patient with methylmalonyl-CoA-mutase deficiency, MMA concentrations in DBSs stored at room temperature for 43 days was 84  approximately 500 times higher than the upper limit of the reference interval. The DBS MMA concentrations of patients with cobalamin C deficiency were between 2.5- and 6- times higher than the upper limit. Table 3-1: Characteristics and dried blood spot (DBS) methylmalonic acid (MMA) concentrations in control samples of children treated for inborn errors of metabolism. Diagnosis Sex Age at sample collection (y) Days of sample at room temperature MMA in 8-mm DBS (pmol) MMA in whole blood (μmol/L) Methylmalonyl-CoA-mutase deficiency  Male 4 43 14,4981 659.01 Cobalamin C deficiency   Male 14 74 128.51 5.841 Female 10 74 179.61 8.161 Male 9 94 75.11 3.411 Propionyl-CoA -carboxylase deficiency Female 16 97 25.0 1.14 1 Value above upper limit of reference interval.  3.5.Discussion To my knowledge, this is the first study to present a reference interval for MMA quantified in DBSs of healthy, term newborns. This will allow for the identification of newborns with subtly elevated DBS MMA concentrations. The reference interval was calculated according to CLSI EP28-A3c using a well-defined sample of n= 160 healthy, term newborns (184). Newborns with DBS MMA concentrations above the upper limit of the reference interval are at increased risk of B-12 deficiency (22,184). Additionally, the effect of DBS storage time at room temperature on MMA concentrations was analyzed to determine the 85  potential impact of storage conditions on the reference interval and ultimately identification of newborns above the reference value. A reference interval of 9.89–29.3 pmol MMA per 8-mm DBS for healthy, term newborns with a mean (95% CI) MMA concentration of 16.8 (15.9; 17.6) pmol in 8-mm spots or 0.764 (0.723; 0.800) µmol/L in whole blood was presented. Turgeon et al. (122) reported mean (range) MMA concentrations for 200 newborn DBSs of 0.6 (0.2–2.0) μmol/L whole blood using LC-MS/MS after derivatization. These findings are comparable to this study, where I reported a mean (range) MMA concentration of 0.76 (0.36–2.3) μmol/L in whole blood. As reported in Chapter 2.4.4 and shown in Figure 2-5, DBS MMA concentrations (n= 10) increase significantly when stored at room temperature for >1 wk. Substantially higher mean MMA concentrations of up to 232 μmol/L whole blood of healthy newborns were reported after prolonged DBS storage at room temperature for 3–104 mo (246). In this study, a small but significant, positive correlation of storage time at room temperature with newborn DBS MMA concentrations was found. DBSs of healthy, term newborns in this study were stored at room temperature for maximum 16 days between sample collection and storage at -80 ºC. Although the increase in DBS MMA concentrations was statistically significant, there was no significant effect on the upper limit of the reference interval.  Thus, this data suggests that storage of newborn DBSs below freezing temperature until analysis is important to achieve reliable results. Yet, DBSs can be stored at room temperature for at least 1 wk without an effect on the DBS MMA concentrations; and for 2 wk with little but non-significant effect on the upper limit of the reference interval. Therefore, shipment at room temperature of DBSs to a 86  reference laboratory is suitable for DBS MMA testing when analyzing DBSs subsequent to receipt or transferring DBSs to appropriate storage conditions (e.g. -80°C freezer). Quantitative assays for DBS MMA have been described using derivatization with subsequent analysis by gas chromatography coupled with mass spectrometry (GC-MS) (229), LC-MS/MS (122,247) or high performance liquid chromatography with fluorescence detector (246). The method reported in Chapter 2 and the assay by La Marca et al. (135) quantify DBS MMA without derivatization using LC-MS/MS. La Marca et al. (135) found DBS MMA concentrations of healthy newborns below their limit of quantification of 1.95 μmol/L whole blood. The low sensitivity and recovery at low DBS MMA concentrations of previously published methods (Table 3-2) has not allowed for a reliable quantification of DBS MMA of healthy newborns. The method presented in Chapter 2, which does not require derivatization, is highly sensitive and has a limit of detection (Table 3-2, bold and Table 2-4) that allows for quantification of MMA in DBSs of healthy adults and newborns. Accordingly, it has the potential to identify newborns with subtly elevated MMA concentration allowing for detection of B-12 deficiency and milder forms of inborn errors of MMA metabolism (64). 87  Table 3-2: Summary of assay performance of different methods for the quantification of methylmalonic acid in newborn dried blood spots using liquid chromatography.1 Method Deriva-tization  Limit of detection Recovery2 (%) Imprecision (CV%)2 Ref. Intra-assay Inter-assay LC-MS/MS  Yes 0.13 μmol/L whole blood 64.7-105.0 5.7-6.9 7.8-9.6 (122) HPLC-FD  Yes 17.4 μmol/L whole blood3 -4 -4 -4 (246) UPLC-MS/MS Yes 0.013 μmol/L whole blood -4 15.9% 11.4 (247) LC-MS/MS  No 1.95 μmol/L whole blood 92.9-106.1 3.5-7.8 3.1-6 (135) LC-MS/MS  No 0.0085 μmol/L whole blood5 92.9-98.0 0.49-1.9 1.2-6.3  1 Ref.: reference; CV: coefficient of variation; LC-MS/MS: liquid chromatography-tandem mass spectrometry; HPLC-FD: high performance liquid chromatography with fluorescence detector: UPLC: ultra-performance liquid chromatography. 2 Ranges across different concentrations.  3 lowest reported concentration.  4 not reported.  5 for comparison purposes, the limit of detection was converted assuming 22 µL whole blood per 8-mm spot.   Methylmalonic acid concentrations are strongly inversely correlated with total B-12 concentrations in newborns (116,241). The reference value for elevated DBS MMA concentrations can be computed as the mean upper limit of the reference interval. In this sample of healthy, term newborns, 4 (2.5%) newborns displayed elevated MMA concentrations above the upper limit, suggestive of B-12 deficiency. Since this project was nested within the BC Newborn Screening program, and samples were de-identified, however, no clinical follow-up data for the newborns or their mothers to confirm these findings of B-12 inadequacy was possible. Results from a study conducted in newborn screening samples in Norway revealed that ~10% of newborns had serum B-12 concentrations below adult cutoffs (150 pmol/L) and ~5% showed metabolic profiles indicative of impaired B-12 function (116). Given that inadequate B-12 status presents with non-specific symptoms in newborns and can lead to infantile B-12 deficiency, testing of neonatal B-12 status has been suggested for inclusion into newborn screening programs (64). Analysis of newborn DBS MMA is a simple initial test that 88  can be followed up with confirmatory testing such as serum total B-12 or holotranscobalamin measurement. With early detection and treatment, long-term sequelae of B-12 deficiency can be prevented. As expected, when comparing DBS MMA concentrations of the healthy newborns, children with inborn errors of B-12 metabolism displayed elevated MMA concentrations. Increases of up to 10,000-fold have been described in untreated newborns with methylmalonic acidemia (122) but much subtler increases can be seen in inherited disorders of cobalamin metabolism. The patient with propionyl-CoA-carboxylase deficiency had DBS MMA concentrations below the upper limit of the reference interval as expected for this inborn error of metabolism (122). Additionally, these results confirm that newborns have higher circulating MMA concentrations compared to adults (116,159,241). DBS MMA concentrations of these 160 healthy newborns were approximately 6-times higher than DBS MMA concentrations of healthy, young adult women (2.93 pmol per 8-mm DBS; n= 96; see also Chapter 2.4.2). Others also reported elevated plasma MMA concentrations in 4-day old newborns (116) and in cord blood samples (149,216) when comparing to adult derived plasma MMA reference values. Reduced renal clearance is a major determinant of circulating MMA concentrations (114) and therefore the physiologic lower renal clearance of neonates and infants (248) may well be the explanation for the increased DBS MMA concentrations observed in this study.  No effect of sex on MMA concentrations and no correlation between birth weight and MMA concentrations were observed in this study, in contrast to findings of previous studies analyzing neonatal serum samples (241,249). However, such effects have so far been reported in single studies only and further research is needed to understand associations between sex or birth weight and B-12 status. 89  3.6.Conclusions As undetected neonatal B-12 inadequacy can progress to severe deficiency in exclusively breast-fed infants, screening neonatal B-12 status is a possible strategy to prevent clinical manifestations. Certain populations with low intakes of animal sourced foods are at increased risk for B-12 deficiency (6).  The strength of this study is that it was nested within the newborn screening program, showing that quantification of MMA is feasible in routinely collected neonatal DBSs and has the potential to test newborns for B-12 deficiency. The data suggest that one has to carefully evaluate sample procedures and storage when setting a reference interval for MMA in newborn DBSs. Storage of DBSs at room temperature for 2 wk leads to a small but significant increase in MMA concentrations. Taking the impact of storage into account, it was feasible to determine a reference value for subtly elevated MMA in newborn DBSs, applicable to the detection of B-12 deficiency or milder forms of inherited errors in MMA metabolism. Yet, further research is needed to confirm B-12 deficiency in newborns with DBS MMA concentrations above the reference range. In practice, elevated DBS MMA would be followed up with further investigation to confirm or rule out B-12 deficiency. Second-tier testing of newborn DBSs has the potential to improve positive predictive values and allow for increased detection of neonatal B-12 deficiency and prevent the associated adverse health outcomes. 90  4. Vitamin B-12 Status and Diagnostic Performance of Vitamin B-12 Biomarkers in Pregnant Women of South Asian and European Ethnicity Residing in Vancouver  4.1.Summary Maternal vitamin B-12 (B-12) status during pregnancy has been inversely associated with adverse health consequences in the mother and offspring. Women of South Asian ethnicity, Canada’s largest ethnic minority, may be at increased risk for B-12 deficiency during pregnancy. Serum total B-12 is the most commonly used biomarker for B-12 status assessment; however, its limitation is low specificity. Holotranscobalamin (holoTC) is the active form of B-12 taken up by the placenta and might thus more closely relate to fetal B-12 status. Circulating holoTC concentration has been suggested to be a more sensitive direct biomarker for B-12 deficiency. To date, no study has assessed the utility of measuring holoTC in pregnant women to determine B-12 deficiency during pregnancy. The objectives of this study were (A) to compare the serum total B-12 concentrations between pregnant women of South Asian and European ethnicity, (B) to determine the diagnostic performance of holoTC compared to total B-12 during pregnancy in detecting functionally B-12 deficient (as indicated by elevated MMA concentrations) pregnant women, and (C) to compute reference values for total B-12 and holoTC, indicative of B-12 deficiency in pregnant women. The study included a retrospective cohort of pregnant women of European (50 %) and South Asian ethnicity and their newborns. Serum samples of healthy women (19–44 y) in their 1st and 2nd trimester [mean (range) gestational age: 11.5 (8.3–13.9) wk (n= 686) and 16.5 (14.9–20.9) wk (n= 748)] and dried blood spot (DBS) samples [mean (range) collection time: 36.8 91  (21.8–143) h postpartum] of their newborns (n= 605; 3.3 % preterm) were obtained from the BC Prenatal Screening Program. Concentrations of total B-12 and holoTC, and of the functional B-12 biomarkers methylmalonic acid (MMA) and total homocysteine (tHcy), and folate were quantified in the maternal serum samples. Offspring functional B-12 status was assessed by determining DBS MMA concentrations. Biomarker concentrations were compared between ethnicities using Wilcoxon rank-sum test. Biomarker performance was evaluated by Spearman’s correlation, ROC analysis, and linear regression. A pregnancy-specific reference value for serum total B-12 in early-mid pregnancy was determined by inflection point analysis. South Asian pregnant women had a significantly lower B-12 status than Europeans, e.g. during the first trimester, mean (95% CI) serum total B-12 concentrations were significantly lower in South Asian compared to European women [189 (180; 199) pmol/L versus 246 (236; 257) pmol/L]. The lower serum total B-12 concentrations in South Asian compared to European women were not reflected in the DBS MMA concentrations of their newborns, which did not differ by ethnicity. Maternal serum concentrations of total B-12 [mean (95%CI): 216 (209; 224) and 200 (193; 206) pmol/L during 1st and 2nd trimester, respectively], holoTC [78.9 (76.4; 81.5) and 74.1 (71.6; 76.6) pmol/L], MMA [131 (127; 135) and 125 (121; 130) nmol/L], and tHcy [4.97 (4.89; 5.06) and 4.23 (4.14; 4.32) µmol/L] significantly decreased with gestational age. In either trimester, the maternal biomarkers were weakly (r= 0.2–0.3) but significantly correlated with neonatal DBS MMA concentrations. Additionally, maternal B-12 biomarker concentrations had only minimal predictive capacity of neonatal circulating MMA concentrations (1.2%–4.5%). 92  During either trimester, there was no difference in the diagnostic capacity of maternal serum total B-12 [AUC (SE): 0.78 (0.03) and 0.80 (0.03)] and serum holoTC [AUC (SE): 0.80 (0.03) and 0.78 (0.03)] concentration in identifying pregnant women with serum MMA concentrations >210 nmol/L. Overall maternal serum total B-12, holoTC, and MMA concentrations had similar, limited predictive capacity to identify mothers who had newborns with DBS MMA concentrations >75th percentile (AUC ~0.60–0.70). A reference value, indicative of functional B-12 deficiency during the 1st and early 2nd trimester, for serum total B-12 was 181 (169; 192) pmol/L based on maternal serum MMA concentrations. In conclusion, pregnant women of South Asian compared to European ethnicity have a substantially lower B-12 status which may render them at increased risk for adverse pregnancy outcomes associated with perinatal B-12 deficiency. Further research is needed to identify predictors and outcomes of the low B-12 status of South Asian pregnant women. Further, maternal serum total B-12 and holoTC performed equally as diagnostic tools in identifying pregnant women with functional B-12 deficiency. However, either maternal biomarker has only minimal capacity in identifying women who may be at risk for having a newborn with functional B-12 deficiency. Lastly, pregnancy-specific cutoffs for B-12 deficiency should be established from novel reference values. 4.2.Introduction Vitamin B-12 (B-12) is involved in DNA synthesis, methylation of biomolecules, and neuron myelination which are crucial processes during fetal development and growth (31,32). Maternal B-12 inadequacy (e.g. serum total B-12 <221 pmol/L) during pregnancy has been 93  associated with a wide array of adverse health outcomes for mother and offspring, including neural tube defects (NTDs), low birth weight, and intra-uterine growth retardation (7,39,54). It has, additionally, been identified as a predictor of neonatal and infant B-12 deficiency, respectively (7,32,35). Adverse health outcomes associated with maternal B-12 inadequacy during pregnancy may be more pronounced in individuals with high intakes of folic acid because of the interrelation of folate and B-12 in the one-carbon metabolism (58,250–252). Given mandatory fortification of flour and the widespread use of prenatal supplements, folic acid intake of reproductive-age women in Canada is high and estimated to exceed the estimated average requirement (EAR) of 320 µg/d Dietary Folate Equivalents (DFEs) (253). Thus, preventing maternal B-12 inadequacy during pregnancy is of emerging interest in Canada (195). Some populations and ethnic groups, including South Asians, with low intakes of animal-source foods are at increased risk for B-12 inadequacy (6). South Asians are the largest ethnic minority in Canada (48) and one of the largest growing ethnic minorities in the United States (254). Prevalence of B-12 deficiency (i.e. serum total B-12 <148 pmol/L) in non-pregnant South Asian populations in Canada and the United States was reported to be higher than in Europeans or other ethnicities in a limited number of studies (169,190,191). Additionally, women of South Asian ethnicity living in Western Countries have a reportedly higher prevalence of newborns with low birth weight and small-for-gestational age compared to women of other ethnicities, including European (255–257). This may, in part, be explained by their potentially low B-12 status. Yet, the prevalence of B-12 inadequacy in pregnant women of South Asian ethnicity residing in Canada is not known.  94  Sensitive and specific assays for determining B-12 status in pregnant women are necessary because of the potentially high prevalence of B-12 inadequacy, especially in certain ethnic minorities, and serious adverse health consequences of B-12 inadequacy during pregnancy. Two direct biomarkers of B-12 status, serum total B-12 and holotranscobalamin (holoTC), are the current clinical standards to determine B-12 deficiency in adults and pregnant women (8,114). HoloTC is the bioactive form of B-12 which is readily available to all tissues (95), including the placenta (88). It has been suggested to be the more sensitive direct indicator of B-12 status in adults (102–105), but this finding is not consistent (99–101). The diagnostic performance of holoTC compared to total B-12 was superior in a large study of outpatients (103), in individuals with previously low B-12 status [plasma methylmalonic acid (MMA) concentrations >280 nmol/L] (104), and in older adults (aged >63 y) (102,105) but was equal in healthy older adults (age ≥60 years) (101) and vegetarians and vegans (99,100). To date, no study has yet assessed the sensitivity and specificity of holoTC or total B-12 in pregnant women. Elevated circulating MMA concentrations are considered to be a sensitive and specific indicator of functional B-12 deficiency, especially in individuals with normal renal function (8,22,114) and newborns (20). MMA concentrations have been reported to remain largely unchanged throughout early pregnancy but increase in late pregnancy (35,87,149,179). As discussed in Chapter 1.2.2, reference values for MMA in healthy non-pregnant adults indicative for B-12 deficiency vary by author from >210 nmol/L to >370 nmol/L (84,180,224). Total B-12 and holoTC concentrations have been reported to decrease during healthy pregnancy due to hemodilution, fetal requirements, and other unknown physiological changes 95  (87,146,148,149). Thus, pregnancy-specific reference values, and eventually established cutoffs, for total B-12 and holoTC may be required for a valid assessment of B-12 status in pregnant women. Given the importance of preventing B-12 inadequacy during pregnancy and a potentially high prevalence of B-12 inadequacy in women of South Asian ethnicity in Canada, tools with high diagnostic accuracy are necessary to determine B-12 status in pregnant women. Thus, the objectives of this study were (A) to compare the serum total B-12 concentrations between pregnant women of South Asian and European ethnicity, (B) to determine the diagnostic performance of holoTC compared to total B-12 during pregnancy in detecting functionally B-12 deficient (as indicated by elevated MMA concentrations) pregnant women, and (C) to compute reference values for total B-12 and holoTC, indicative of B-12 deficiency in pregnant women. 4.3.Materials and Methods 4.3.1. Study Design and Setting This retrospective cohort study utilized residual serum and DBS samples and demographic and health, which had been routinely collected by the BC Prenatal Screening Program (BC Children’s Hospital, Vancouver, Canada). Maternal samples were selected from serum samples collected during prenatal genetic screening [BC Prenatal Genetic Screening Program (258)]. The program is offered free of charge and is recommended for all pregnant women residing in BC (Canada) (259). Participation is entirely voluntary and it is estimated that 50% of all pregnant women residing in BC participate in the program (H. Vallance, personal communication). The primary objective of the program is to screen for pregnancies 96  affected by chromosomal disorders [e.g. Down Syndrome (Trisomy 21), Trisomy 18] and open NTDs (258). As such, the women participating in the program and, thus, the present study may be older, have experienced previous pregnancy complications, or may be more anxious, educated or health conscious than the general population (259,260). Further details are discussed in the limitations of the study (Chapter 4.5.4). The screening involves 2 visits at approximately 11±2 wk (1st trimester) and 17±2 wk (2nd trimester) of gestation. Non-fasting venous blood samples and demographic and other health-related information are recorded at each visit. Neonatal DBS samples of newborns of mothers whose serum samples had been included in the study were retrieved from the BC Newborn Screening Program (BC Children’s Hospital, Vancouver, Canada) (261). DBS samples are routinely collected from every newborn in BC (Canada) and sent to the program for analyses. Specimens are stored for 10 years following routine testing and can be utilized for deidentified research, in keeping with the mission of the newborn screening program, unless parents or legal guardians request removal or destruction of the stored sample (261). Given the retrospective access to residuals of routinely collected samples, nature of the sample analyses, and legal regulations, collection of informed consent was waived. The Clinical Research Ethics Board of The University of British Columbia (Vancouver, Canada) and the Newborn Screening Research Review Committee at BC Children’s Hospital (Vancouver, Canada) approved this study. 97  4.3.2. Sample Size The sample size calculation was based on the 1st objective to determine a difference in B-12 status (serum total B-12 concentrations) between pregnant women of South Asian and European ethnicity. The study aimed to retrieve sets of 2 serum samples (1st trimester and 2nd trimester) collected from 600 women of South Asian (n= 300) and European (n= 300) ethnicity during pregnancy. A set consisted of 1 sample collected at 11±2 wk (1st trimester) and 1 sample collected at 17±2 wk (2nd trimester) of gestation. This sample size allowed for the detection of a difference in serum total B-12 concentrations between pregnant women of South Asian and European ethnicity of 40 pmol/L with a power (1-β) of 0.80 at a confidence level (α) of 0.05 (Equation 4-1 and Table 4-1). In addition to maternal samples, DBS samples of newborns (n= 600) of women in the study were retrieved after birth. To allow for an attrition rate of 17% (due to neonatal sample not eligible for the study, insufficient residual DBS sample, or newborn born outside BC) samples of 700 pregnant women (n= 350 South Asian and 350 European, respectively) were included in the study. Sample collection was continued until sufficient sets of maternal samples had been retrieved (N= 1,400 from 700 women).  Equation 4-1: Sample size calculation. 𝒏𝒏 = 𝟐𝟐𝝈𝝈𝟐𝟐×(𝒁𝒁𝜷𝜷 + 𝒁𝒁𝜶𝜶 𝟐𝟐⁄ )𝟐𝟐(𝝁𝝁𝟏𝟏 − 𝝁𝝁𝟐𝟐)𝟐𝟐   98  Table 4-1: Statistical variables for sample size calculation. Statistical Variable  Power (1-β) 0.80 Confidence level (α) 0.05 Difference in total B-12 (μ1-µ2) (pmol/L) 401 Standard deviation (ϭ) (pmol/L) 1751 Sample size (n)2 302 Effect size (Cohen’s d) >0.2 1 based on Quay et al. and Milman et al. (169,179). 2 per group. 4.3.3. Study Sample and Data Collection Maternal samples Maternal non-fasting serum samples were included in the study if the women had identified themselves as being either of South Asian or European ethnicity and were aged 19–45 y old. Additionally, only samples collected within the Lower Mainland (BC, Canada) were retrieved for the study. Samples of women who had a multiple gestation, a pregnancy after in-vitro fertilization or steroid use, diabetes mellitus (I/II), a history of smoking, or a positive screen for a chromosomal disorder or open NTD in this or a past pregnancy were excluded from the study. Information on maternal age, self-identified ethnicity, and gestational age at blood collection (estimated by crown-rump-length measured using ultrasound) was obtained from medical charts filled during the prenatal screening visit. Neonatal samples All available neonatal DBS samples matched to maternal serum samples were retrieved. Samples were excluded from the analyses if newborns were screened positive for an inborn error of metabolism or received total parenteral nutrition or blood transfusion. Neonatal information on sex, birth weight, preterm birth, and time of DBS collection was obtained from 99  medical charts filled during birth and DBS sample collection. Gestational age at birth was estimated from gestational age and date at maternal sample collection and birth date as noted in the charts. Sample processing Whole maternal blood samples were routinely collected into serum separator tubes. Serum was separated from whole blood within 24 hours of sample collection by centrifugation at 1890 x g for 5 min at 4°C. Serum samples were transferred to polypropylene tubes, stored at 4°C and transferred to -80°C within 4 days. For this study, samples were retrieved from the -80°C storage, thawed on ice, and separated into aliquots (one aliquot per analyte) allowing for deidentification. Subsequently, aliquots were stored at -80°C until specific biomarker analyses. Neonatal DBS samples had been stored at -20°C after completion of routine analyses. Upon completion of sample retrieval for all eligible newborns, DBS samples were thawed at room temperature (~18–21°C), de-identified, and transferred to -80°C until further analyses. Freeze-thaw cycles at which DBSs are at room temperature (~18–21°C) for ≤24 h do not affect DBS MMA concentration (data not shown). DBS MMA concentrations, however, increase during prolonged storage (>14 d) at room temperature (Figure 2-5). Thus, the time that samples had spent at room temperature (~18–21°C) during routine processing was recorded. DBS samples that had been stored at room temperature for >14 d were excluded from the analyses (n= 41).  100  4.3.4. Biochemical Analyses of Serum Total Vitamin B-12, Holotranscobalamin, Methylmalonic Acid, Total Homocysteine, and Folate Preface Maternal serum samples were analyzed sequentially for the B-12 biomarkers total B-12, holoTC, MMA, and tHcy as well as folate. The order of the analytes (volume of aliquot) was: total B-12 (120 µL), holoTC (200 µL), MMA (50 µL), folate (10 µL), tHcy (50 µL). If the sample volume was insufficient for an aliquot, the analyte was not quantified in the respective sample. Neonatal DBS samples were analyzed singly for DBS MMA.  Serum total vitamin B-12 and holotranscobalamin quantification by automated immunoassays Serum total B-12 and holoTC were quantified by fully automated immunoassays (Access by Beckman Coulter Inc. and Architect by Abbott Technologies, respectively) at the pathology laboratories at BC Children’s Hospital (Vancouver, Canada) and St. Paul’s Hospital (Vancouver, Canada), respectively. The inter-assay CV for 4 serum total B-12 control samples (mean c: 93.6 pmol/L; 245 pmol/L; 335 pmol/L; 407 pmol/L) ranged from 2.4% to 7.1% (analyzed over 4 mo); the control samples provided by the manufacturer for holoTC were within the control range of 15.5±1.5 pmol/L and 46.5±3.5 pmol/L (analyzed over 3 d). The holoTC assay had an upper limit of 128 pmol/L.   101  Serum and dried blood spot methylmalonic acid quantification by liquid chromatography-tandem mass spectrometry Methylmalonic acid in serum and DBSs was determined as previously described (Chapter 2.3). In brief, MMA was extracted from 8-mm DBS punches. After sample clean-up, MMA in serum and DBS extract was quantified by stable isotope dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS). The inter-assay CV for an in-house control sample was 7% (n= 19) for serum and 15% (n= 8) for DBS MMA. The intra-assay CV (n= 2) was <5% for all analyses.  2-Methylcitric acid (MCA) was analyzed in maternal serum samples only. It was quantified simultaneously with MMA by LC-MS/MS with d3-MMA as the internal standard. MCA exists as 4 enantiomers: RS, SR, RR, and SS. The RS- and RR-enantiomers are found in human samples (119). The MCA standard (Sigma-Aldrich) used for this assay contained all 4 enantiomers, 2 of which eluted simultaneously (SR with RS and SS with RR, respectively) using the chromatographic conditions presented in Table 2-1. The 2 enantiomers present in the maternal serum samples (RS and RR) eluted separately. The results are presented as sum of all enantiomers and are referred to as MCA. The inter-assay CV for an in-house control MCA sample was 14% (n= 19). Serum total homocysteine quantification by liquid chromatography-tandem mass spectrometry Serum tHcy was quantified using isotope dilution LC-MS/MS based on a method outlined by Friesen et al. (262). In brief, after reduction with dithiothreitol and protein precipitation, samples were injected into an LC system (Agilent 1260, Agilent Technologies). 102  Compounds were separated by a normal phase column (Fortis H2O, 2.1 x 150 mm, 5µm, Fortis Technologies). The mobile phase consisted of (A) 0.2% heptafluorobutyric acid in water and (B) 0.2% heptafluorobutyric acid in acetonitrile using a gradient run [A:B 95:5 (v/v) to 20:80 (v/v)]. The affluent was directed into an MS/MS system (API4000, SCIEX Pte). Serum tHcy was quantified with a 7-point calibration curve (1.14–114.48 µmol/L) made using L-homocysteine (Sigma Aldrich) as calibrator and d4-homocysteine (Cambridge Isotope Laboratories) as internal standard. The inter-assay CV for an in-house control tHcy sample was 9.4% (n= 17). Two external quality control samples manufactured by Clinchek 23082 and IRIS Technologies International were quantified with every analysis and were within their acceptable ranges of 9.0±1.8 µmol/L and 25.9±5.1 µmol/L, respectively. Serum folate quantification by microbiological assay Serum folate was analyzed using the microbiological assay according to the method developed by O’Broin and Kelleher (1992) and Molloy and Scott (1997) (263,264). The assay was performed in 96-well plates and using the chloramphenicol-resistant Lactobacillus rhamnosus (ATCC 27773). All sample and standard preparation was carried out under amber light. Thawed serum samples were diluted 1:80 with 1% ascorbic acid and prepared to 2 final dilutions of 1:1,600 and 1:3,200 in 0.5% sodium ascorbate. Ten-point (0–0.87 nmol/L) calibration curves were made using 5-methyltetrahydrofolate [(6S)-5-methyl-5,6,7,8-tetrahydropteroyl-L-glutamic acid, sodium salt; Merck Eprova] (265) and computed by quadratic regression. Samples and calibrators were mixed with inoculated broth [Difco folic acid casei medium, Becton-Dickinson; 1:2 (v/v)] and incubated at 37°C for 42 h. The Westgard Rules were applied to test performance and ensure integrity of the assay (266). The National Institute for Biological Standards and Control reference material 95/528 (NIBSC 95/528) and 103  an in-house serum control sample were included with each of the 22 runs. The analyses yielded folate contents of 28.3 nmol/L (13 ng/mL) (inter-assay CV: 8.8%) for the NIBSC 95/528 control sample (manufacturer value: 13 ng/mL), and of 42.8 nmol/L (inter-assay CV: 10.1%:) for the in-house serum control sample. 4.3.5. Classification of Vitamin B-12 and Folate Status Classification of vitamin B-12 status To date, there are no established cutoffs to define B-12 status in pregnancy for any of the four available B-12 biomarkers. Pregnant women were, thus, classified using non-pregnant adult reference values or cutoffs to allow for comparison with previous research (Table 4-2).  From here forth, B-12 status of pregnant women will be referred to as B-12 deficiency, suboptimal B-12 status, and B-12 adequacy. B-12 inadequacy refers to combined B-12 deficiency and suboptimal B-12 status. It is commonly accepted that serum total B-12 concentrations <148 pmol/L or serum holoTC concentrations <35 pmol/L indicate B-12 deficiency in non-pregnant adults (84,86,175). Serum total B-12 concentrations <221 pmol/L and serum holoTC concentrations <55 pmol/L during early pregnancy (<28 d) have been associated with an increased risk for NTD-affected pregnancies (38,39). Given the lack of pregnancy-specific cutoffs and the use of non-pregnant adult cutoffs in the present study, the results should be interpreted with caution. 104  Table 4-2: Cutoffs and reference values for serum concentrations of total vitamin B-12 (total B-12), holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine (tHcy) used to classify pregnant women’s B-12 status into B-12 deficiency, suboptimal status, and adequacy. Biomarker Deficiency Suboptimal status Adequacy Total B-12 (pmol/L) <148 148– <221 ≥221 HoloTC (pmol/L) <35 35– <55 ≥55 MMA (nmol/L) >3701 210– >3701 ≤210 tHcy (µmol/L) >13 N/A ≤13 1with 2-methylcitric acid concentrations < MMA concentrations.  Circulating MMA concentrations are strongly confounded by renal function. However, measurement of MCA allows controlling for renal function (119). Elevated and mildly elevated MMA concentrations were defined as serum MMA concentrations >370 nmol/L (183), which has been described as “generally agreed on cutoff” (84) in adults, and >210 nmol/L, the 95th percentile of the NHANES population with serum total B-12 concentrations >50th percentile (84), respectively, with MCA concentrations<MMA concentrations (119). Neonatal functional B-12 status was assessed by DBS MMA concentration. Functional B-12 deficiency was defined as DBS MMA >29.3 pmol/8-mm punch (Figure 3-1).   Classification of folate and total homocysteine status Serum tHcy concentrations >13 µmol/L have been used to define elevated tHcy concentrations in pregnant women and populations with folic acid fortification (84,146,267). In Canada, mandatory fortification of flour with folic acid has been in place since 1998 (268). Folate deficiency was defined as serum folate concentration <6.8 nmol/L and suboptimal status as 6.8–13.4 nmol/L, as proposed by the World Health Organization; serum folate concentrations >45.3 nmol/L were considered elevated (84,269). 105  4.3.6. Statistical Analyses Descriptive statistics All statistical analyses were performed in Stata 14.2 (Stata Corp LP) for Windows 10 (Microsoft Corp.), with the level of significance set at P< 0.05. Normality of data was tested visually [quantile plot (270)] and using the Shapiro-Wilk test (236). Unless stated otherwise, the data are presented as geometric mean with range (minimum to maximum) or 95% confidence interval (95% CI). Differences in serum biomarker concentrations between women of European ethnicity and women of South Asian ethnicity were determined by Wilcoxon rank-sum test; differences in prevalence were determined by Pearson’s chi-squared test. The correlation between birthweight or gestational age at birth and maternal serum biomarker concentrations was explored by Spearman’s rank correlation. Differences in serum biomarker concentrations between women who had preterm or low birth weight newborns and women who had term or normal birthweight newborns were explored by Wilcoxon rank-sum test. Analytical bias of the holotranscobalamin assay The holoTC assay had an upper limit of 128 pmol/L (83.5th percentile of the overall study population). Individuals with serum holoTC concentrations >128 pmol/L were excluded from most non-descriptive statistical analyses (linear models), i.e. mixed effects model of maternal biomarker concentrations and gestational age, logistic regression, and receiver operating characteristics (ROC) analysis.    106  Prediction of maternal biomarker concentrations by gestational age  The prediction of maternal biomarker concentrations by gestational age was determined by linear mixed effects modeling adjusted for repeated measures. The model was performed on natural log-transformed data to achieve a more normalized distribution of the residuals. The coefficient β (95% CI) was reported. The mean change in maternal serum total B-12 and holoTC concentration was calculated as stated in Equation 4-2. For better comparison between the biomarkers the change relative to the 1st trimester concentration (%) in maternal serum total B-12 and holoTC concentration was further estimated as stated in Equation 4-3. Equation 4-2: Calculation of the change per week (pmol/L) in maternal serum total vitamin B-12 and holotranscobalamin concentration. 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑐𝑐ℎ𝑚𝑚𝑚𝑚𝑎𝑎𝑚𝑚 𝑝𝑝𝑚𝑚𝑝𝑝 𝑤𝑤𝑚𝑚𝑚𝑚𝑤𝑤 (𝑝𝑝𝑚𝑚𝑝𝑝𝑝𝑝𝐿𝐿)=  𝑏𝑏𝑏𝑏𝑝𝑝𝑚𝑚𝑚𝑚𝑝𝑝𝑤𝑤𝑚𝑚𝑝𝑝 𝑐𝑐𝑝𝑝𝑚𝑚𝑐𝑐𝑚𝑚𝑚𝑚𝑐𝑐𝑝𝑝𝑚𝑚𝑐𝑐𝑏𝑏𝑝𝑝𝑚𝑚 (𝑝𝑝𝑚𝑚𝑝𝑝𝑝𝑝𝐿𝐿 )2𝑛𝑛𝑛𝑛 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 − 𝑏𝑏𝑏𝑏𝑝𝑝𝑚𝑚𝑚𝑚𝑝𝑝𝑤𝑤𝑚𝑚𝑝𝑝 𝑐𝑐𝑝𝑝𝑚𝑚𝑐𝑐𝑚𝑚𝑚𝑚𝑐𝑐𝑝𝑝𝑚𝑚𝑐𝑐𝑏𝑏𝑝𝑝𝑚𝑚 (𝑝𝑝𝑚𝑚𝑝𝑝𝑝𝑝𝐿𝐿 )1𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑎𝑎𝑚𝑚𝑔𝑔𝑐𝑐𝑚𝑚𝑐𝑐𝑏𝑏𝑝𝑝𝑚𝑚𝑚𝑚𝑝𝑝 𝑚𝑚𝑎𝑎𝑚𝑚 (𝑤𝑤𝑤𝑤)2𝑛𝑛𝑛𝑛 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 − 𝑎𝑎𝑚𝑚𝑔𝑔𝑐𝑐𝑚𝑚𝑐𝑐𝑏𝑏𝑝𝑝𝑚𝑚𝑚𝑚𝑝𝑝 𝑚𝑚𝑎𝑎𝑚𝑚 (𝑤𝑤𝑤𝑤)1𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡    Equation 4-3: Calculation of the mean change per week (%) relative to the 1st trimester concentration in maternal serum total vitamin B-12 and holotranscobalamin concentration. 𝑝𝑝𝑚𝑚𝑝𝑝𝑚𝑚𝑐𝑐𝑏𝑏𝑟𝑟𝑚𝑚 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑐𝑐ℎ𝑚𝑚𝑚𝑚𝑎𝑎𝑚𝑚 𝑝𝑝𝑚𝑚𝑝𝑝 𝑤𝑤𝑚𝑚𝑚𝑚𝑤𝑤 (%)= 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑑𝑑𝑚𝑚𝑐𝑐𝑝𝑝𝑚𝑚𝑚𝑚𝑔𝑔𝑚𝑚 𝑝𝑝𝑚𝑚𝑝𝑝 𝑤𝑤𝑚𝑚𝑚𝑚𝑤𝑤 (𝑝𝑝𝑚𝑚𝑝𝑝𝑝𝑝𝐿𝐿 )𝑏𝑏𝑏𝑏𝑝𝑝𝑚𝑚𝑚𝑚𝑝𝑝𝑤𝑤𝑚𝑚𝑝𝑝 𝑐𝑐𝑝𝑝𝑚𝑚𝑐𝑐𝑚𝑚𝑚𝑚𝑐𝑐𝑝𝑝𝑚𝑚𝑐𝑐𝑏𝑏𝑝𝑝𝑚𝑚(𝑝𝑝𝑚𝑚𝑝𝑝𝑝𝑝𝐿𝐿 )1𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ∗ 100  107  Correlations of maternal and neonatal biomarker concentrations Maternal serum concentrations of total B-12, holoTC, MMA, tHcy, and folate in pregnant women were correlated by Spearman’s rank correlation. The same correlation method was used to relate the B-12 biomarker concentrations in pregnant women and DBS MMA concentration in the newborns.  Linear regression analyses Multiple linear regression models were fitted to identify the predictive capacity of maternal serum concentrations of total B-12, holoTC, and MMA, respectively, on neonatal DBS MMA concentrations (natural log-transformed). The models were adjusted for confounders, i.e., maternal ethnicity (South Asian), maternal age (y), newborn sex (female), newborn age at birth (h), birth weight (g), and gestational age at birth (wk). Three series of models were constructed: (Model 1) only confounding factors were included; (Models 2) 1st trimester serum total B-12, holoTC, and MMA concentrations were individually added to Model 1; (Models 3) 2nd trimester serum total B-12, holoTC, and MMA concentrations were individually added to Model 1. Models were compared using the Akaike Information Criterion (AIC) (271). Receiver operating characteristic analyses Receiver operating characteristic curves were constructed to compare the diagnostic performance of maternal serum total B-12, serum holoTC, and serum MMA concentrations in discriminating pregnant women with and without impaired functional B-12 status. Two series of ROC curves were computed: Women were classified as having ‘impaired functional B-12 108  status’, when (i) their serum MMA concentrations were >210 nmol/L and MCA<MMA with or without elevated serum tHcy concentrations [given the low tHcy concentrations in Canadian pregnant women due to the high folate intake (194)]; (ii) the DBS MMA concentrations of their newborns was >75th percentile. The latter was chosen to have sufficient pregnant women classified as having ‘impaired functional B-12 status’, i.e. relatively lower B-12 status, to allow for ROC analyses. It is, however, recognized that the DBS MMA concentrations of the newborns are within the normal range. Neither of the 2 criteria may truly reflect B-12 inadequacy in the pregnant women, which is discussed further in the limitations of this study (Chapter 4.5.4). Sensitivity analyses were performed using more conservative criteria, i.e. (i) maternal serum MMA concentrations >370 nmol/L, and (ii) neonatal DBS MMA concentrations >29.3 pmol/8-mm spot. The ROC curves were compared by testing the equality of the area under the curve (AUC). The sensitivity and specificity of commonly used cutoffs and reference values of total B-12 and holoTC were estimated. Comparison of maternal serum methylmalonic acid and neonatal dried blood spot methylmalonic acid concentrations after classifying pregnant women’s vitamin B-12 status using different direct biomarkers and cutoffs (or reference values if no cutoff available) The utility of commonly used serum total B-12 and holoTC cutoffs and reference values to identify functional B-12 deficiency in pregnant women was compared. This was achieved by comparing (i) serum MMA concentrations and (ii) matched neonatal DBS MMA concentrations of women with serum total B-12 concentration <148 pmol/L to women with serum holoTC concentration <35 pmol/L. The test was repeated comparing women with serum total B-12 concentrations <221 pmol/L to women with serum holoTC concentrations <55 pmol/L. MMA concentrations were compared using Wilcoxon rank-sum test. 109  Determination of reference values Lastly, piecewise linear regression was applied to describe the relationship between maternal serum MMA concentration and maternal serum total B-12 and serum holoTC concentration, respectively. To reduce heteroscedasticity and skewness of the data, natural log-transformed maternal serum MMA concentration was chosen as the dependent variable. The model assumed a linear-linear relationship with 1 inflection point in which the slopes of the 2 fitted lines changed significantly. The inflection point was estimated using the ‘nl hockey estimation’ add-on (nlhockey.ado by Mark Lunt) for Stata 14.2. 4.4.Results 4.4.1. Subject Characteristics To meet the sampling goal of the study, samples and data of 748 women (aged 19–44 y) were collected (Figure 4-1). Of these women, 686 (92%) attended both prenatal screening visits, and serum samples from the 1st and 2nd trimester were retrieved; 62 (8%) women did not attend the 1st screening visit due to unknown reasons. The mean (range) gestational age at the 2 study time points was 11.5 wk (8.3–13.9) and 16.5 wk (14.9–20.0), respectively (Table 4-3). There were no significant differences in maternal characteristics between pregnant women of European and South Asian ethnicity (Table 4-3). 110   Figure 4-1: Flow diagram of sample collection and analyses, and reasons for missing samples.  * Differences in number of biochemical analyses results from insufficient serum volume.  $ DBS samples not received by the Newborn Screening laboratory likely due to unsuccessful pregnancy, missing maternal personal health number on newborn screening card, or delivery of newborn outside of the province of British Columbia. B-12, vitamin B-12; holoTC, holotranscobalamin; MMA, methylmalonic acid; tHcy, total homocysteine; DBS, dried blood spot.  111  Table 4-3: Subject characteristics [mean (range) or prevalence] of pregnant women and their newborns. Characteristic  All European South Asian P-value1 Maternal      n 751 376 375 -  Age (y) 31.0 (19-44) 30.9 (19-40) 31.1 (20-44) 0.8  Gestational age (wk) (1st trimester visit) 11.5 (8.3-13.9) 11.5 (8.3-13.9) 11.4 (8.3-13.9) 0.3  Gestational age (wk) (2nd trimester visit) 16.5 (14.9-20.9) 16.5 (14.9-20.9) 16.6 (15.0-20.9) 0.3 Neonatal      n 536 284 252 -  Sex (male; %) 49.2 48.9 49.4 0.8  Birth weight (g) 3369 (1207-4770) 3487 (2198-4770) 3233 (1207-4475) 0.0009  Low birth weight (<2500 g; %) 1.9 0.62 3.3 <0.0001  Gestational age at birth (wk) 38.8 (27.6-42.9) 38.9 (27.6-42.9) 38.7 (28.3-42.9) 0.01  Preterm birth (<37 wk of gestation; %) 3.3 2.5 4.2 0.3  Time of sample collection (h postpartum) 34.5 (21.8-144) 36.7 (23.7-143) 32.2 (21.8-106) <0.0001  Long transit (>14 d; %) 7.6 3.2 12.7 <0.0001 1 P-value indicating difference between women of European and South Asian ethnicity (self-reported) as tested by Wilcoxon rank-sum test (continuous variables) or chi-squared test (categorical variables); -: not applicable; NS: not significant.  DBS samples of 605 newborns were retrieved for this study (Figure 4-1); 143 DBS samples were not collected because of insufficient residual DBS sample (n= 59), storage of newborn screening card at room temperature for > 4 weeks (n= 12), or inability to link to a newborn specimen (n= 72) (Figure 1). Inability to link a maternal sample to a DBS sample may be due to unsuccessful pregnancy, missing maternal personal health number on newborn screening card, failure of a DBS card to be collected and/or received in the laboratory, or because the newborn was delivered outside of the province of British Columbia. Newborn characteristics are summarized in Table 4-3. Fifty neonatal DBS samples (8.3%) were 112  excluded from the MMA analyses due to their extended storage at room temperature (> 14 d).  Women of South Asian ethnicity had significantly more newborns with low birth weight (<2500 g) compared to women of European ethnicity. Additionally, newborns of South Asian women were born at a significantly lower gestational age, although this was not reflected when analyzing preterm birth. Forty-one neonatal DBS samples (7.6%) were excluded from the MMA analyses due to their extended storage at room temperature (>14 d; long transit); more DBS samples of newborns of South Asian women (n= 32) than of newborns of European women (n= 9) were excluded from the analyses. 4.4.2. Maternal and Neonatal Vitamin B-12 and Folate Status Vitamin B-12 biomarker concentrations were quantified in at least 300 samples from women of each South Asian and European ethnicity (in total n= 600) per time point, i.e., the calculated target sample size, except for tHcy concentrations (Figure 4-1). The results for the pregnant women are summarized in Table 4-4.113  Table 4-4: Mean (95% CI) serum concentrations of total vitamin B-12 (B-12), holotranscobalamin (holoTC), methylmalonic acid (MMA), total homocysteine (tHcy), and folate of women of European and South Asian ethnicity residing in the Lower Mainland (BC, Canada) and prevalence of inadequate and deficient vitamin status during the 1st and 2nd trimester of pregnancy.1 Biomarker 1st Trimester 2nd Trimester  All European South Asian P-value2 All European South Asian P-value2 Total B-12 (pmol/L) 216 (209; 224) n= 684 246 (236; 257)  n= 351 189 (180; 199) n= 333 <0.0001 200 (193; 206) n= 746 226 (216; 236) n= 377 176 (168; 185) n= 369 <0.0001  <221 pmol/L [% (n)] 50.6 (346) 39.4 (138) 62.5 (208) <0.0001 57.9 (433) 48.4 (182) 68.0 (251) <0.0001  <148 pmol/L [% (n)] 20.2 (137) 10.6 (37) 30.1 (100) <0.0001 25.4 (189) 15.3 (57) 35.8 (132)  <0.0001 HoloTC (pmol/L) 78.9 (76.4; 81.5) n= 673 85.1 (81.9; 88.3) n= 340 73.1 (69.4; 77.1) n= 333 0.0003 74.1 (71.6; 76.6) n= 652 78.6 (75.5; 81.9) n= 325 69.8 (66.2; 73.6) n= 327 0.0090  <55 pmol/L [% (n)] 19.5 (131) 12.0 (41) 27.1 (90) <0.0001 25.0 (163) 19.6 (64) 30.3 (99) 0.0010  <35 pmol/L [% (n)] 5.3 (36) 1.5 (5) 9.2 (31) <0.0001 5.3 (35) 2.5 (8) 8.2 (27) 0.0010 MMA (nmol/L) 131 (127; 135) n= 662 120 (115; 124) n= 327 143 (137; 151) n= 335 <0.0001 125 (121; 130) n= 697 114 (109; 119) n= 345 138 (130; 146) n= 352 <0.0001  >210 nmol/L [% (n)]3 12.7 (85) 5.3 (18) 20.3 (67) <0.0001 11.8 (83) 5.5 (19)  18.2 (64) <0.0001  >370 nmol/L [% (n)]3 6.0 (41) 2.1 (7) 10.2 (34) <0.0001 7.0 (49) 3.2 (11)  10.8 (38)  <0.0001 tHcy (µmol/L) 4.97 (4.89; 5.06) n= 631 4.98 (4.88; 5.10) n= 338 4.97 (4.84; 5.10) n= 293 0.5 4.23 (4.14; 4.32) n= 513 4.34 (4.20; 4.78) n= 240 4.14 (4.02; 4.27) n= 273 0.1  >13 µmol/L [% (n)] 0 0 0 ND 0 0 0.36 (1) 0.3 Folate (nmol/L)  65.6 (63.7; 67.6) n= 665 66.1 (63.5; 68.8) n= 334 65.1 (62.4; 68.0) n= 331 0.5 67.7 (65.7; 69.6) n= 696 67.3 (64.6; 70.1) n= 348 68.4 (65.7; 71.2) n= 348 0.4  >45.3 nmol/L [%(n)]4 85.7 (580) 86.0 (295) 85.3 (285) 0.8 87.3 (607) 85.8 (195) 89.2 (313) 0.1  <13.4 nmol/L [%(n)]4 0.15 (1) 0 0.3 (1) 0.3 0 0 0 ND  <6.8 nmol/L [%(n)]4 0 0 0 ND 0 0 0 ND 1 Serum samples were non-fasting. 2 P-value indicating difference between women of European and South Asian ethnicity (self-reported) as tested by Wilcoxon rank-sum test (biomarker concentrations) or chi-squared test (prevalence); ND: not determined due to very low prevalence. 114  3 in combination with serum 2-methylcitric acid (MCA) concentration < serum MMA concentration. 4 indicative of folate deficiency (<6.8 nmol/L), folate inadequacy (<13.4 nmol/L), and elevated circulating folate concentrations (>45.3 nmol/L).115  Pregnant women of South Asian compared to European ethnicity had significantly lower serum total B-12 and serum holoTC concentrations and a significantly higher serum MMA concentration, during the 1st and 2nd trimester of pregnancy (Table 4-4). The effect size of the difference was largest for serum total B-12 (Cohen’s d: 0.52 and 0.45, respectively) and smallest for holoTC during the 1st and 2nd trimester (Cohen’s d: 0.28 and 0.20, respectively). Significantly more women of South Asian ethnicity were classified as B-12 deficient and B-12 inadequate, respectively, as indicated by serum total B-12 (<148 and <221 pmol/L), serum holoTC (<35 and <55 pmol/L) or serum MMA (>370 and >210 nmol/L) concentration, than women of European ethnicity at both time points in the study. There was no difference in serum tHcy and folate concentrations between pregnant women of South Asian and European ethnicity.  The prevalence of B-12 deficiency among pregnant women in this study differed substantially depending on biomarker and time point of sample collection. It ranged from 5.3% (serum holoTC <35 pmol/L, both time points) to 25.4% (serum total B-12 <148 pmol/L, 2nd trimester) (Table 4-4). More than half of all women had inadequate B-12 status as indicated by total serum B-12 <221 pmol/L at both visits. However, only ≤25% were classified as having inadequate B-12 status at both visits when using serum holoTC <55 pmol/L as indicator. The prevalence of elevated serum MMA concentrations (>370 nmol/L and MCA<MMA) and mildly elevated serum MMA concentrations (>210 nmol/L and MCA<MMA) was ~6.5% and ~12%, respectively. Functional B-12 deficiency was not reflected in elevated serum tHcy concentrations (>13 µmol/L, ~0%). No woman displayed folate deficiency (serum folate <6.8 nmol/L) but the prevalence of elevated serum folate concentrations (>45.3 nmol/L) was 116  high (>80%) at both time points. However, it has to be noted that the samples were collected at non-fasting conditions and serum folate concentration undergoes high diurnal changes (85). Considering that 22 µL whole blood creates a DBS with 8-mm in diameter (data not shown), newborns had a mean (95% CI) MMA concentration of 713 (700; 736) nmol/L whole blood. Neonatal DBS MMA concentration was not significantly different between South Asians and Europeans (Table 4-5). The overall prevalence of B-12 deficiency in the newborns (DBS MMA >29.3 pmol/punch) was ~2% with no difference between newborns of women of South Asian and European ethnicity (Table 4-5). Table 4-5: Neonatal dried blood spot methylmalonic acid (DBS MMA) concentrations of newborns of women of European and South Asian ethnicity. Biomarker All European South Asian P-value1 DBS MMA (pmol/8-mm punch) 15.7 (15.3; 16.2) n= 555 15.3 (14.8; 15.8) n= 298 16.2 (15.5; 16.9) n= 257 0.4  >75th percentile [% (n)] N/A 25.1 (75)  24.1 (62)  0.8  >29.3 pmol/punch [% (n)] 2.2 (12) 1.3 (4)  3.1 (8)  0.2 1 P-value indicating difference between newborns of women of European and South Asian ethnicity (self-reported) as tested by Wilcoxon rank-sum test (biomarker concentrations) or chi-squared test (prevalence).  Maternal serum total B-12 concentrations during 1st and 2nd trimester were positively correlated with birthweight (r= 0.083; P= 0.04 and r= 0.082; P= 0.03, respectively); maternal serum holoTC, MMA, or tHcy concentrations during either trimester were not significantly correlated with birthweight. Gestational age at birth was positively correlated with maternal 1st and 2nd trimester serum total B-12 (r= 0.081; P= 0.04 and r= 0.10; P=0.01, respectively) and serum holoTC (r= 0.089; P= 0.03 and r= 0.095; P= 0.02) concentrations; it was not correlated with maternal serum MMA or serum tHcy concentrations during either trimester. There was 117  no significant difference in any B-12 biomarker concentration between women who had low birthweight or preterm newborns and women who had normal birthweight or term newborns. Gestational age was a significant negative predictor of maternal serum total B-12, serum holoTC (pregnant women with holoTC concentrations >128 pmol/L excluded), serum MMA, and serum tHcy concentrations (all natural log-transformed; Figure 4-2). Assuming a linear relationship, the mean (95% CI) decrease per week in serum total B-12 and holoTC concentrations was -3.5 (-5.3; -1.8) pmol/L (n= 668) and -0.77 (-1.1; -0.40) pmol/L (n= 474), respectively. This equals to a mean (95% CI) weekly change in serum total B-12 and holoTC concentrations of -0.33 (-1.04; 0.38) % and -0.32 (-0.91; 0.26) %, respectively, relative to the 1st trimester concentration. MMA concentrations appeared to remain unchanged [mean (95% CI) weekly change: -0.19 (-1.2; 0.9) nmol/L; n= 618]. Serum total homocysteine concentrations changed by a mean (95% CI) of -0.15 (-0.18; -0.11) μmol/L per week (n= 387) or by -0.02 (-0.03; -0.02) % relative to the 1st trimester concentration.  Maternal serum folate concentrations (data not shown) were not predicted by gestational age.118   Figure 4-2: Changes in concentrations (natural log-transformed) of serum total vitamin B-12 (B-12), holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine (tHcy) with gestational age in pregnant women.  Red line indicates linear prediction: Coefficient (β) was computed using a mixed effects model; P represents the corresponding likelihood-ratio.119  4.4.3. Correlations of Maternal and Neonatal Biomarker Concentrations All maternal biomarker concentrations were significantly correlated except for serum folate with each of serum MMA and tHcy concentrations at both time points in the study (Table 4-6); however stronger with MMA than tHcy. There was no significant correlation between serum folate and tHcy concentrations.  Table 4-6: Spearman’s rank correlation coefficients (ρ) between maternal serum metabolite concentrations at 1st and 2nd trimester and neonatal dried blood spot methylmalonic acid (DBS MMA) concentrations at birth.1   Total B-12 HoloTC MMA tHcy DBS MMA 1st  Trimester Total B-12     -0.15 (525) HoloTC 0.64  (669)    -0.22 (410) MMA -0.41  (669) 0.44  (659)   0.20  (512) tHcy -0.29 (626) -0.34  (617) 0.29 (563)  0.01  (480) Folate 0.09  (670) 0.17 (659) -0.02 (659) 0.02 (623) -0.08  (514) 2nd  Trimester Total B-12     -0.20 (554) HoloTC 0.70 (656)    -0.22 (439) MMA -0.39 (700) -0.44 (615)   0.28 (521) tHcy - 0.29 (516) -0.33  (456) 0.36 (514)  0.10 (381) Folate 0.14 (700) 0.14 (611) 0.02 (653) -0.02 (479) -0.05 (516) 1 B-12: vitamin B12; holoTC: holotranscobalamin; MMA: methylmalonic acid; tHcy: total homocysteine; bold numbers indicate statistical significance (P< 0.05); number in brackets indicates sample size; 1st trimester visit mean (range) gestational age: 11.5 (8.3–13.9) wk; 2nd trimester visit mean (range) gestational age: 16.5 (14.9–20.9) wk.  Maternal serum total B-12 and serum holoTC concentrations appeared to be equally correlated with either functional biomarker (MMA or tHcy) concentration; however, stronger 120  with MMA than tHcy. There was no significant correlation between maternal serum folate and tHcy concentrations. After adjusting for gestational age, maternal serum total B-12 and serum holoTC concentrations remained significantly inversely correlated with DBS MMA. Maternal serum holoTC concentration appeared to be equally correlated with neonatal DBS MMA as maternal serum total B-12 concentration. 4.4.4. Receiver Operating Characteristics Curves of Vitamin B-12 Biomarkers to Discriminate Pregnant Women with Impaired Functional Vitamin B-12 Status To further compare the performance of maternal serum total B-12 and serum holoTC concentrations as a diagnostic tool to discriminate between pregnant women with or without ‘impaired functional B-12 status’, 2 series of ROC curves were constructed [reference variable: (i) maternal serum MMA concentration >210 nmol/L; (ii) neonatal DBS MMA concentrations >75th percentile] (Figure 4-3). Given the limitations of the study, that only limited data on functional outcomes in the pregnant women or their newborns were determined, sensitivity analyses were conducted using the “generally agreed on cutoff” (84) in adults and the upper limit of the reference interval for DBS MMA (Figure 3-1) [reference variable: (i) maternal serum MMA concentration >370 nmol/L; (ii) neonatal DBS MMA >29.3 pmol/8-mm DBS]. 121   Figure 4-3: Receiver operating characteristics (ROC) curves for maternal serum total vitamin B-12 (total B-12; black solid line), holotranscobalamin (holoTC; black dashed line), and methylmalonic acid (MMA; grey dotted line) concentrations to discriminate women with A and B (top row): serum MMA >210 nmol/L (and 2-methylcitric acid<MMA); or C and D (bottom row): having a newborn with DBS MMA concentrations >75th percentile.  Maternal samples were collected at 2 time points during pregnancy: 1st trimester (A and C, left column) mean (range) gestational age: 11.5 (8.3–13.9) wk; and 2nd trimester (B and D, right column) mean (range) gestational age: 16.5 (14.9–20.9) wk.   First, pregnant women in this study were classified as having ‘impaired functional B-12 status’ when they had mildly elevated serum MMA concentrations (>210 nmol/L). Results of 1 individual with serum MMA >210 nmol/L and MCA>MMA were excluded from the analyses because of suspected renal abnormalities (119). Pregnant women with serum holoTC concentrations above the upper limit of the assay (128 pmol/L; 83.5th percentile of the study 122  population) were excluded from the analyses. A total of 520 and 529 pregnant women had complete data (i.e. serum total B-12, holoTC and MMA results to be included in the analyses) at the 1st and 2nd trimester of pregnancy, respectively. Of these women 80 (15.4%; A) in the 1st trimester and 74 (14.0%; B) in the 2nd trimester were classified as having ‘impaired functional B-12 status’. The AUCs for maternal serum total B-12 and holoTC were significant (>0.5) and did not differ at either time point (Figure 4-3, top row). The sensitivity and specificity of the commonly used cutoff for serum total B-12 concentration of <148 pmol/L were 63% and 86% during the 1st trimester and 66% and 81% during the 2nd trimester; the sensitivity and specificity of serum holoTC concentration <35 pmol/L were 29% and 98% during the 1st trimester and 23% and 97% during the 2nd trimester. Sensitivity analyses revealed no differences when classifying pregnant women using serum MMA concentrations >370 nmol/L to classify women as B-12 deficient [AUCs (SE): total B-12: 0.88 (0.03); holoTC: 0.86 (0.04) and total B-12: 0.80 (0.04); holoTC: 0.82 (0.03) for the 1st and 2nd trimester, respectively]. In a second ROC analysis, women were classified as having ‘impaired functional B-12 status’ when the neonatal DBS MMA concentrations of their newborns were above the 75th percentile (18.5 pmol/8-mm spot). Of a total of 392 newborns whose mothers had complete data, 102 (26.0%) had DBS MMA concentrations >75th percentile. The AUCs for maternal serum total B-12, serum holoTC, and serum MMA concentrations collected during the 1st or 2nd trimester were small (<0.8) but significant (>0.5) (Figure 4-3, bottom row). In the 1st trimester, the AUC of holoTC was significantly larger than the AUC of total B-12. However, there was no difference in the 2nd trimester. Sensitivity analyses revealed similar results when using neonatal DBS MMA >29.3 pmol/8-mm DBS as reference variable. Although there was no significant difference between holoTC and total B-12 in the 1st 123  trimester, there was a tendency for a difference [AUCs (SE): total B-12: 0.56 (0.10); holoTC: 0.65 (0.12); MMA: 0.63 (0.11) and total B-12: 0.62 (0.10); holoTC: 0.63 (0.12); MMA: 0.71 (0.08) for the 1st and 2nd trimester, respectively].  Comparison of Maternal Vitamin B-12 Biomarkers to Predict Functional Vitamin B-12 Status in Newborns To compare maternal serum total B-12, serum holoTC, and serum MMA concentrations as predictors of neonatal circulating MMA concentrations, 2 series of linear regression models were constructed with neonatal DBS MMA concentrations (natural log-transformed) as outcome variable (Table 4-7).  To allow for comparison of the models, only mother-newborn dyads with complete data sets, i.e. maternal serum total B-12, holoTC, MMA, and neonatal DBS MMA results, were included (women with holoTC concentrations >128 pmol/L excluded). A total of 392 dyads were included in the analyses. Maternal and neonatal confounders, i.e., maternal age, maternal ethnicity, neonatal sex, neonatal age at sample collection, birth weight, and gestational age at birth, alone described 8.5% (R2 = 0.085) of the variation in neonatal DBS MMA concentrations (Model 1). Ethnicity was not a significant predictor of neonatal DBS MMA concentration; newborn age at sample collection and gestational age at birth were the only significant predictors of neonatal DBS MMA concentration. However, it should be noted that either model only explained ~10% in the variation of neonatal DBS MMA concentration.124  Table 4-7: Coefficients (95% CI) of linear regression models predicting neonatal dried blood spot methylmalonic acid concentrations (natural log-transformed).   1st T r i m e s t e r 2nd T r i m e s t e r  Model 1: Confounders Model 2A: Total B-12 Model 2B: HoloTC Model 2C: MMA Model 3A: Total B-12 Model 3B: HoloTC Model 3C: MMA β (95% CI) N/A -0.0004  (-0.0007; -0.0001) P= 0.009 -0.002  (-0.004; -0.001) P< 0.001 0.0008  (0.0004; 0.001) P< 0.001 -0.0007  (-0.001; -0.0003) P< 0.001 -0.003  (-0.004; -0.002) P< 0.001 0.0009  (0.0006; 0.001) P< 0.001 R2 0.085 0.10 0.13 0.13  0.097  0.12 0.13  AIC 0.43 0.41 0.38 0.39 0.42 0.40 0.38 1 B-12: vitamin B12; holoTC: holotranscobalamin; MMA: methylmalonic acid; AIC: Akaike Information Criterion. The models were adjusted for confounders: maternal age (y), maternal ethnicity (South Asian), newborn sex (female), newborn age at sample collection (h), birth weight (g), and gestational age at birth (wk). Maternal samples were collected at 2 time points during pregnancy: 1st trimester mean (range) gestational age: 11.5 (8.3–13.9) wk; and 2nd trimester mean (range) gestational age: 16.5 (14.9–20.9) wk.     125  First, maternal serum total B-12, serum holoTC, and serum MMA concentrations collected during the 1st trimester of pregnancy were analyzed in separate models adjusted for maternal and neonatal confounders (Models 2). Adding maternal 1st trimester serum total B-12 concentration into the model (Model 2A) explained an additional 1.5% of the variation compared to the confounders alone (Model 1). An additional 4.5% of the variation was described after adding maternal serum holoTC concentration into the model (Model 2B). When modelling biomarker concentrations collected during the 2nd trimester (Models 3), maternal serum total B-12 concentrations explained an additional 1.2% (Model 3A) and serum holoTC concentration an additional 3.5% of the variation (Model 3B). Maternal serum MMA concentrations described an additional 4.5% (Model 2C and 3C). When comparing the models, adding any biomarker into the model resulted in better quality models than the confounders alone. In each trimester, serum total B-12 resulted in the model of the worst fit (AIC: 0.41 and 0.42, respectively), although the difference to serum holoTC and MMA concentration was only minimal (AIC: 0.38–0.40). Although there was an increase in the predictive capacity (R2) and quality (AIC) of each model, the factor change [exp(coefficient)] of neonatal DBS MMA concentrations of each unit increase in biomarker concentrations was low (~1). Further, ethnicity was not a significant predictor of neonatal DBS MMA concentration; preterm birth was the only significant predictor of neonatal DBS MMA concentration. However, it should be noted that both models failed to explain ~95% in the variation of neonatal DBS MMA concentration. Additionally, the following analyses were performed to further evaluate the capacity of maternal serum total B-12 and serum holoTC concentrations and their commonly used cutoffs 126  and reference values in identifying pregnant women with functional B-12 deficiency. The mean serum MMA concentration of pregnant women with serum total B-12 concentration <148 pmol/L was compared to that of women with holoTC concentration <35 pmol/L (Figure 4-4, top row). The analyses were repeated comparing mean MMA concentration of women with serum total B-12 concentration <221 pmol/L to those with serum holoTC concentration <55 pmol/L. Mean serum MMA concentration was significantly higher in women classified as B-12 deficient using holoTC concentration compared to when using serum total B-12 concentration at either time point. A similar tendency (although not statistically significant) was observed when comparing the DBS MMA concentration of the newborns, whose mothers’ B-12 status had been classified using either serum total B-12 or holoTC concentration (Figure 4-4, bottom row). Similar results were observed when comparing maternal MMA concentrations or DBS MMA concentrations of newborns of pregnant women who were classified as B-12 adequate using either total B-12 or holoTC (data not shown). Pregnant women with serum total B-12 concentrations ≥221 pmol/L had significantly lower serum MMA concentrations than pregnant women with serum holoTC concentrations ≥55 pmol/L (P< 0.05). There was no difference between their newborns. Further, there was no difference in neonatal DBS MMA concentrations between newborns whose mothers had been classified as B-12 deficient based on serum total B-12 (<148 pmol/L) or holoTC (<35 pmol/L) and newborns whose mothers had been classified as B-12 inadequate based on serum total B-12 (<221 pmol/L) and holoTC (<55 pmol/L).  127   Figure 4-4: Comparison of (top row): the geometric mean (95% CI) serum methylmalonic acid (MMA) concentrations of pregnant women classified as vitamin B-12 (B-12) deficient and having inadequate B-12 status, respectively, using serum total B-12 concentrations (<148 and <221 pmol/L, respectively) and serum holotranscobalamin (holoTC) concentrations (<35 and <55 pmol/L, respectively); and (bottom row): the geometric mean dried blood spot (DBS) MMA concentrations of their newborns.  P-values indicate statistical significance as tested by Wilcoxon rank-sum test. Maternal samples were collected at 2 time points during pregnancy: 1st trimester mean (range) gestational age: 11.5 (8.3–13.9) wk; 2nd trimester mean (range) gestational age: 16.5 (14.9–20.9) wk.  128  4.4.5. Calculation of Methylmalonic Acid-Derived Reference Values for Serum Total Vitamin B-12 and Serum Holotranscobalamin Concentrations Inflection point analysis was used to derive pregnancy-specific reference values for serum total B-12 and holoTC concentrations (Figure 4-5). The analyses were performed using maternal serum MMA concentration (natural log-transformed) as the dependent variable in a linear-linear model. There was no difference between the inflection points (95% CI) for serum total B-12 computed with the results from the 1st trimester and the 2nd trimester visit [181 (165; 197) pmol/L and 182 (165; 199) pmol/L, respectively]. The inflection point computed from the combined data (both time points) was 181 (169; 192) pmol/L for total B-12. No inflection point could be computed for holoTC, given the mostly linear relationship between serum MMA (natural log-transformed) and serum holoTC concentration (Figure 4-5; bottom row). 129   Figure 4-5: Plot of maternal serum total vitamin B-12 (B-12) concentration (top row) and maternal serum holotranscobalamin (HoloTC) concentration (bottom row) against maternal serum methylmalonic acid (MMA) concentration (natural log-transformed) with locally weighted smoothing line (LOESS; red line) during the 1st trimester [A; mean (range) gestational age: 11.5 (8.3–13.9) wk; n= 680] and the 2nd trimester [B; mean (range) gestational age: 16.5 (14.9–20.9) wk; n= 702].  4.5. Discussion The following section discusses the results of this study of B-12 status in 748 pregnant women of European and South Asian ethnicity and their newborns. First, the prevalence of B-12 deficiency and inadequate B-12 status in the population is described with a focus on the 130  difference between pregnant women of European and South Asian ethnicity as well as the discrepancy between biomarkers and cutoffs. Given that no pregnancy-specific cutoffs have yet been established, non-pregnant adult cutoffs (or reference values) were used for comparison purposes. Further, the utility of the direct biomarkers total B-12 and holoTC to diagnose pregnant women with B-12 deficiency are explored with the overall goal to identify the most appropriate diagnostic tool to determine B-12 status in pregnant women. Lastly, the need for and use of pregnancy-specific reference values is emphasized and discussed using results from this study.  4.5.1. Prevalence of Vitamin B-12 Deficiency in Pregnant Women of South Asian and European Ethnicity and Their Newborns Residing in Metro Vancouver In the present study of 748 pregnant women, South Asian compared to European women had a significantly lower B-12 status, as indicated by serum total B-12, holoTC, and MMA concentrations during early pregnancy. The difference in mean serum total B-12 concentration between South Asian and European women in the 1st and 2nd trimester was substantial (57 pmol/L and 50 pmol/L, respectively). Additionally, South Asian ethnicity was a significant negative predictor of maternal B-12 status, as assessed by total B-12, holoTC, and MMA concentrations. During 1st and 2nd trimester, the prevalence of pregnant women who were classified as overtly B-12 deficient (total B-12 <148 pmol/L and MMA >370 pmol/L) was ten and six times higher, respectively, among South Asian (~9% and ~8%, respectively) than European women (~1% at both time points ). Thus, findings from this study in Vancouver suggest that South Asian women compared to European women have a markedly lower B-12 status during the 1st and 2nd trimester of pregnancy. 131  Neonatal B-12 status was assessed in the present study using DBS MMA concentrations. Newborns had a mean (95% CI) DBS MMA concentration of 713 (700; 736) nmol/L whole blood, with no difference between ethnicities. These results are comparable to the previously reported findings in ethnically diverse, healthy, term newborns in Vancouver, Canada (Table 1-6), and in healthy newborns in the US (122). The overall prevalence of B-12 deficiency in newborns, as indicated by DBS MMA concentration above the reference interval (29.3 pmol/ 8-mm punch, Figure 3-1), was ~2%. Hence, the prevalence of elevated MMA concentrations (>370 nmol/L) was higher among pregnant women (6–7%) compared with their newborns. Further, ethnicity was not a predictor of newborn DBS MMA.As such, the observed difference in B-12 status between pregnant women of South Asian and European ethnicity is not reflect in their newborns. It appears that newborns may be protected in utero from developing B-12 deficiency; a hypothesis, which will be further discussed below. The 10% to 15% prevalence of European women with total B-12 concentrations <148 pmol/L during was comparable to previous reports of B-12 status during early pregnancy in Canada with 5% to 17% of predominantly European pregnant women being classified as B-12 deficient (total B-12 <148 pmol/L) (156,181). A recent study in Vancouver found a 16% prevalence in European women (n= 150) at 20–35 weeks of gestation (193). In the present study, 1·5% and 2·5% of European women had holoTC concentrations <35 pmol/L during 1st and 2nd trimester, respectively. Similarly, the prevalence of holoTC concentrations <35 pmol/L in the Alberta Pregnancy Outcomes and Nutrition study was negligible.  Studies from rural and urban India reported a substantially higher prevalence of approximately 50% to 70% of pregnant women having total B-12 concentrations <150 pmol/L during early pregnancy (35,59,272); one study from Nepal found a 28% prevalence during the 1st trimester (57,273). 132  Seventy to 95% of pregnant women in India had MMA concentrations >260 nmol/L (35,59). The 30% to 36% and especially the 20% prevalence of South Asian pregnant women with total B-12 concentrations <148 pmol/L and mildly elevated MMA concentrations (>210 nmol/L), respectively, in the present study is somewhat lower than findings from the Indian subcontinent. Yet, it is at least twice of the prevalence found in European pregnant women in the present study or other Canadian cohorts. Low intakes of animal-source foods, low socioeconomic status, and a lower prevalence of supplement use have been suggested as predictors of B-12 deficiency in South Asians (189,274,275). No dietary or demographic data were available in the present retrospective study to determine predictors of the reported differences in B-12 status. Additionally, some genetic factors may influence B-12 biomarker concentrations, independent of B-12 intake (124). Variants in the FUT2 gene have been identified as predictors of low serum total B-12 concentrations in South Asians (276) and have been suggested to explain part of the association between low serum total B-12 concentration and obesity (277). An increased prevalence of obesity has been described in South Asian populations, including South Asian pregnant women in the UK (275,278). Future research is warranted to identify dietary, socioeconomic, and genetic predictors of the observed lower B-12 status in pregnant women of South Asian ethnicity living in Canada to allow for targeted interventions.  Overall, the prevalence of pregnant women classified as B-12 deficient depends on the biomarker and cutoffs used; using total B-12 <148 pmol/L resulted in an at least four-times higher prevalence of pregnant women classified as B-12 deficient in the present study than any other indicator. This tendency was also reflected in the prevalence of pregnant women classified as B-12 inadequate. I acknowledge that physiological changes during pregnancy may 133  impact biomarker concentrations independent of B-12 status, and pregnancy-specific cutoffs are currently lacking. Yet, infants of South Asian mothers who had serum total B-12 concentrations <148 pmol/L at ~11 wk of gestation were reported to have a significantly higher (~27%) HOMA-IR at age 6–8 years, indicative of a potentially increased risk for developing insulin resistance (57). Further, Indian mothers who had low total B-12 concentration <148 pmol/L during 1st and 2nd trimester [median (IQR): 115 (104; 125) pmol/L and 112 (99; 122) pmol/L, respectively] had increased odds [OR (95% CI): 5.98 (1.72; 20.74) and 9.28 (2.90; 29.68), respectively] of intra-uterine growth retardation than mothers with higher total B-12 concentrations [median (IQR): 224 (206; 268) pmol/L and 210 (177; 217) pmol/L, respectively] (54). These findings were confirmed in a recent meta-analysis. Mothers with serum total B-12 concentrations <148 pmol/L during pregnancy were at increased risk for having a newborn with low birth weight [<2500 g; adjusted risk ratio (95% CI): 1.15 (1.01; 1.31)] or preterm birth [<37 wk; adjusted risk ratio (95% CI): 1.21 (0.99; 1.49)] (60). Preliminary correlational analyses revealed a weak positive association between maternal serum total B-12 concentrations during the 1st and 2nd trimester and newborn birthweight as well as between maternal serum total B-12 and serum holoTC concentrations and gestational age at birth. However, no difference in B-12 status was observed between women who had low birthweight or preterm newborns compared to women who had healthy newborns. The latter observation may be explained by the low prevalence of low birthweight and preterm birth of 1.9% and 3.3%, respectively, in the study leading to a small sample size and power to detect a difference. Thus, although further research is needed to develop and evaluate pregnancy-specific cutoffs for B-12 deficiency, low B-12 status and especially total B-12 concentrations 134  <148 pmol/L have been associated with adverse pregnancy outcomes in South Asians and other ethnicities. Inadequate maternal B-12 status at <28 days of gestation has been associated with an increased risk for NTDs (38,39). The prevalence of NTDs in Canada decreased substantially by 46% to 4.1/10,000 births since the introduction of mandatory food fortification with folic acid in 1998 (279). Since then, B-12 has been discussed as a major determinant of NTDs occurring in Canada (38). It has been estimated that 34% of all NTDs occurring in Canada may be attributed to inadequate maternal B-12 status (38). Ray et al. (38) found a serum holoTC concentration <55 pmol/L was associated with a 3-fold increase in risk of having a NTD-affected pregnancy. Molloy et al. (39) reported that women with serum total B-12 concentrations <221 pmol/L commencing pregnancy have a significantly increased risk (twice) for NTD-affected pregnancies. In the present sample ~20% and ~50% of women, respectively, had serum total B-12 and holoTC concentrations associated with an increased risk for NTD-affected pregnancies during the 1st trimester of pregnancy. However folate status, the strongest predictor of NTD defects (36), was high in the pregnant women in the present study. Given that at least 20% of the women in this study displayed a B-12 status associated with an increased risk of NTDs during the 1st trimester of pregnancy, a high prevalence of Canadian women may have a B-12 status during early pregnancy that has been associated with an increased risk for NTD-affected pregnancies. None of the women in this study had elevated tHcy concentrations or folate deficiency. Given mandatory folic acid fortification and the high prevalence of prenatal supplement use, folate status in Canadian pregnant women (156,181,221), is high and elevated tHcy is found rarely (194). More than 85% of the pregnant women in the present study had elevated 135  circulating folate concentrations (>45.3 pmol/L); however, these results need to be interpreted with caution because serum samples were collected in the non-fasting state, which substantially affects circulating folate concentrations (85). Plasma folate concentrations have been reported to increase by more than 30-times after ingestion of 500 µg folic acid (280). Prenatal supplements typically contain ~1 mg folic acid. Others reported similar high folate concentrations in non-fasting pregnant women in Canada (221). Thus, the present study contributes to the body of evidence that folate status in pregnant women in Canada is high, while B-12 status is low (156,195,253,268). In summary, findings of this study support that there is an overall high prevalence of pregnant women in Canada classified as B-12 deficient. Yet given the use of non-pregnant adult cutoffs (or reference values), these results should be interpreted with caution. Women of South Asian compared to European ethnicity residing in British Columbia have a substantially lower B-12 status. Given that B-12 is important for fetal development and that South Asians are Canada’s largest ethnic minority, future research is warranted to address the potential causes for lower B-12 status in South Asian pregnant women and to understand whether the lower maternal B-12 status impacts infant B-12 status and stores over time.  4.5.2. Comparison of Total Vitamin B-12 and Holotranscobalamin as Indicator of Functional Vitamin B-12 Status in Pregnant Women This pregnancy cohort study with 748 pregnant women aimed to test the performance of different maternal B-12 biomarkers to predict neonatal B-12 status and diagnose deficiency. The study used banked maternal serum and matched newborn DBS samples from existing public health screening programs. Prior studies have used cord blood samples. The use of 136  newborn DBS samples, collected between 24–48 hours after birth, has several advantages compared to cord blood. The unique composition of cord blood with a high concentration of stem cells, challenges in reliable sample collection, and the tight connection to the maternal metabolism may affect biomarker concentrations in cord blood samples. DBS samples collected between 24–48 hours after birth are much more reflective of neonatal metabolism (161). In the present study, maternal B-12 biomarker concentrations explained only minimal changes in neonatal DBS MMA concentrations (1.2%–3.9%) and were only weakly correlated with neonatal DBS MMA concentrations (Spearman’s ρ: 0.2–0.3). Others who have described the relationship between maternal B-12 status (i.e. total B-12, holoTC, and MMA concentrations) during early pregnancy and cord blood MMA concentrations (149,156) or other markers of neonatal B-12 status in DBSs, e.g., acylcarnitines (281), found similarly weak or non-significant correlations. Further, neither maternal B-12 biomarker appeared to have a strong diagnostic utility in identifying women at risk of having a newborn with impaired functional B-12 status (DBS MMA >75th percentile or >29.3 pmol/8-mm DBS) in the present study. Thus, the findings of this study suggest that maternal B-12 status during 1st and 2nd trimester is a poor predictor and indicator of neonatal functional B-12 status at birth.  An overexpression of the holoTC-transporter CD320 in placental tissue (88) and significantly higher total B-12 concentrations in cord blood (149,156,159) and amniotic fluid (282) compared with maternal total B-12 concentrations suggest that the fetus may be protected in utero from developing B-12 deficiency through a preferential unidirectional transport of B-12 in pregnant women. This may, in part, explain the weak correlation, small predictive capacity, and poor diagnostic utility of maternal B-12 biomarker concentrations during early pregnancy of neonatal functional B-12 status at birth observed in the present study. As such, 137  the results of the present study suggest that newborns of women with low B-12 status may not present with B-12 deficiency at birth but rather develop symptoms of B12 deficiency over time as has previously been discussed (32,61,62). Breastmilk of B-12 deficient mothers may supply insufficient B-12 to the infant during the exclusive breastfeeding period (0–6 mo postpartum) rendering infants at risk for developing B-12 deficiency (35,212). This is of particular importance given the evidence that even mild B12 deficiency during infancy can cause impaired motor and cognitive development (79) that improves with supplementation (27,28,73,77).  Maternal serum total B-12 and serum holoTC concentration had the same capacity to diagnose pregnant women who displayed mildly elevated and elevated serum MMA concentrations (>210 nmol/L and >370 nmol/L, respectively). The AUC for either biomarker was ~0.8 to identify women with mildly elevated MMA concentrations during early pregnancy. Studies in non-pregnant adults (103,104) and older adults (102,105) have revealed similar AUCs for total B-12, however, slightly higher AUCs for holoTC of 0.8–0.9 with holoTC being the significantly better indicator of B-12 deficiency. Two studies in non-pregnant adults (99,100) found no difference between the biomarkers, which may be explained by the small sample sizes (n< 200). Miller et al (101) also reported no difference between total B-12 and holoTC in older adults (≥60 y), however, the cut-offs (MMA > 350 nmol/L with tHcy >13 μmol/L) the authors used for defining B-12 deficiency were lower than in other studies of older adults (MMA >750 nmol/L) (102). In the present study, pregnant women were categorized as having ‘impaired functional B-12 status’ when having mildly elevated serum MMA concentrations (>210 nmol/L) and sensitivity analyses revealing similar results were performed with elevated serum MMA concentrations (>370 nmol/L). I acknowledge that no 138  pregnancy-specific cut-offs for MMA have yet been established and the present study does not have any follow-up data, confirming B-12 deficiency in pregnant women or their newborns. Also, disturbances in circulating MMA concentrations in pregnant women and cord blood independent of B-12 status have recently been described (124). As such, B-12 status might have been misclassified. Yet, the diagnosis of B-12 deficiency in pregnant women and newborns is challenging as symptoms are non-specific (32,62). Because folate is the major determinant of tHcy concentration, and Canadian pregnant women were shown to have high intakes of folic acid from prenatal supplements and fortified foods (221,283), and only one of the pregnant women had elevated serum tHcy concentrations (>13 µmol/L), MMA was used as sole indicator of impaired functional B-12 status. In summary, findings from this study suggest that, in contrast to non-pregnant adults and older adults, serum total B-12 and holoTC perform equally well in identifying pregnant women with impaired functional B-12 status.  No consensus has yet been reached on pregnancy-specific cutoffs for total B-12 and holoTC concentrations; cutoffs and reference values for the general population are often used in pregnant women (156,181,221,222). The most commonly used serum total B-12 cutoff to identify individuals with B-12 deficiency is <148 pmol/L (114). In the present study, the sensitivity of this cutoff was ~60% and the specificity ~80%. The reference value for holoTC to indicate B-12 deficiency, i.e., <35 pmol/L, had a substantially lower sensitivity (~30%) but higher specificity (~90%). Additionally, women with serum holoTC concentrations <35 pmol/L in the 1st and 2nd trimester had significantly higher mean (95% CI) serum MMA concentrations [313 (252; 374) nmol/L and 266 (208; 325) nmol/L, respectively] than those with serum total B-12 concentrations <148 pmol/L [219 (195; 245) nmol/L and 171 (159; 183) nmol/L, respectively]. A similar tendency was also reflected in the DBS MMA 139  concentrations of their newborns, although not statistically significant. This suggests that women classified as B-12 deficient using serum holoTC concentration and the reference value of <35 pmol/L have a significantly lower intracellular B-12 status than women classified as B-12 deficient using serum total B-12 <148 pmol/L. Thus, total B-12 <148 pmol/L is a more sensitive, while holoTC <35 pmol/L is a more specific indicator of B-12 deficiency in pregnant women. Yet, further research is needed to develop and evaluate pregnancy-specific reference values and establish cutoffs. The decrease of circulating biomarker concentrations due to hemodilution or other physiological changes emphasizes the need for pregnancy-specific cutoffs. In the present study, maternal serum total B-12 concentration decreased significantly during pregnancy, which has been well established in previous studies (147–149). Additionally, maternal serum holoTC concentration decreased at a similar rate (-0.33% per week) as maternal serum total B-12 concentration. A decrease in maternal serum MMA concentration during 1st and 2nd trimester of pregnancy was only observed on log-transformed data. Circulating holoTC and MMA concentrations have so far been described to remain largely unchanged during early pregnancy (87,146,148,151) with MMA concentrations increasing at the end of pregnancy (87,146). The larger sample size in the present study that allowed for detecting minimal changes in biomarker concentrations might explain some of the discrepancies to previous studies. It does not support the hypothesis that the observed decrease in total B-12 during pregnancy may be attributed to a shift in B-12 binding proteins and a decrease of haptocorrin-bound B-12 only (87,151). Given that pregnancy-related physiological changes, such as hemodilution, may lead to a natural decrease in biomarker concentrations, pregnancy-specific cut-offs are required for many nutrients (118). Findings from the present study confirm the 140  need for pregnancy-specific cut-offs for B-12 biomarker concentrations, especially for total B-12 and holoTC. In conclusions, findings from the present study revealed that maternal B-12 biomarker concentrations, i.e. total B-12, holoTC, and MMA, during early pregnancy are equally poor predictors of neonatal functional B-12 status, assessed by DBS MMA concentrations, at birth. Yet, maternal serum total B-12 and holoTC are equally strong in diagnosing pregnant women with elevated serum MMA concentrations. More research is warranted to establish pregnancy-specific cut-offs for maternal B-12 biomarker concentrations. 4.5.3. Pregnancy-Specific Reference Values for Total Vitamin B-12 and Holotranscobalamin to Assess Vitamin B-12 Status The physiological changes occurring during pregnancy require the modeling of pregnancy-specific cutoffs for many nutrients (12,118). The most important factor may be the natural decrease in biomarker concentrations due to hemodilution or other physiological changes. In the present study, maternal serum total B-12 and holoTC concentrations decreased between 1st and 2nd trimester of pregnancy, as discussed above.  Using an MMA-derived inflection point, a reference value of <181 pmol/L was computed for serum total B-12 concentration during the first 2 trimesters of pregnancy. No reference value could be computed for serum holoTC concentration given the mostly linear relationship between serum MMA (natural log-transformed) and holoTC concentrations in this sample of pregnant women. The linearity may in part be explained by the upper limit of the holoTC assay (128 pmol/L; 82.5th percentile of the population) which could lead to a distortion of the curve describing the relationship. Several approaches have been established to develop 141  and evaluate cutoffs, including inflection point analysis, ROC analyses and reference intervals (12,111). The advantage of an inflection point analysis is that the computed reference value directly correlates with a physiological outcome (12), in the present study with impaired intracellular B-12 status in the mother. However, it has to be noted that the segmented linear regression with 1 inflection point used in this study may not represent the best-fitted model (111). Bailey et al. (111) found that a linear splines model with 2 inflection points and 3 categories of B-12 status (i.e. deficiency, suboptimal status, adequacy) best describes the relationship between serum total B-12 and MMA concentrations in the NHANES 1999-2004 population (n= 12,683; non-pregnant adults ≥19 y). Given the much smaller sample size of the present study, such an approach was not possible. Thus, although the suggested reference value in the present study reflects use of a well-established approach to classify women during early pregnancy into B-12 deficient and adequate, a larger data set might reveal a 3rd category of B-12 status in pregnant women. Pregnancy-specific reference intervals (179) and biomarker cutoffs based on NTD risk (38,39) have been previously suggested. Milman et al. (179) computed a lower limit (2.5th percentile) of 96 pmol/L at 18 wk of gestation for plasma total B-12 from a healthy population of 434 healthy pregnant women (non-supplement users). This is substantially lower than the reference value suggested in the present study. The discrepancy may partially be explained by the methods that were used to calculate the reference values. Similar trends were observed in non-pregnant adults, where the inflection point analysis revealed higher reference values of 194 pmol/L (111) and 334 pmol/L (115) than limits of reference intervals (148 pmol/L) (177). The reference value for serum total B-12 computed in the present study, i.e., <181 pmol/L at `11–16 wk of gestation, is surprisingly in agreement with the serum total B-12 concentration 142  of <184 pmol/L that Molloy et al. (39) found to be associated with an increased risk of NTD-affected pregnancies at 20 wk of gestation. Future research is needed to determine the specificity and sensitivity as well as clinical performance of the suggested reference value to diagnose women at risk for adverse pregnancy outcomes associated with B-12 deficiency. In summary, this study contributes to the body of evidence about B-12 biomarker concentrations and their reference values during healthy pregnancy. Using adult cutoffs or reference values may not be valid in pregnant women given pregnancy-specific physiological changes. A newly suggested reference value for total B-12 represents the concentration after which MMA concentration increases rapidly. This reference value may be more clinically meaningful in identifying pregnant women with B-12 deficiency. Future research is needed to test the diagnostic performance and validity of the reference value determined by inflection point analysis and establish a cutoff. 4.5.4. Strengths and Limitations This study of 748 pregnant women of South Asian and European ethnicity was nested within the public health system in BC, Canada. Samples were accessed through the Perinatal Screening Program and consent was waived in these mother-infant pairs. The strengths of this study design include the longitudinal approach and the lack of consent bias. Participants were also not biased towards the nutrient of interest. However, recruitment was limited in that only pregnant women who chose to participate in the prenatal screening in BC could be included in the study. Given that prenatal screening is designed to detect pregnancies affected by chromosomal abnormalities and open NTDs, the women participating in the program and, thus, in this study might be older, have experienced previous pregnancy complications, or may be 143  more anxious, educated or health conscious than the general population (259,260). Metcalfe et al. (259), however, observed no significant differences in prevalence of preterm births or low birth weight in women attending prenatal screening visits in Canada. Birth outcomes with significant differences between women attending and not attending prenatal screening visits, i.e., previous NTD-affected pregnancies, were an exclusion criterion for this study. Thus, although women were not actively recruited, the study likely has a sampling bias and may not be representative of the general population. Further strengths of this study include a very large sample size, multiple time points, and a portfolio of biomarkers especially all common direct and functional biomarker of B-12 status (114). This large sample size allowed detecting even subtle changes and differences. Additionally, a comprehensive assessment of B-12 status was possible, given the multiple B-12 biomarkers, as has been recommended by experts (126,130). In summary, this study provides a comprehensive evaluation of B-12 status in pregnant women of South Asian and European ethnicity in Canada. The main limitation of this retrospective study is the lack of data on dietary intake and prenatal vitamin supplement use. One may thus not draw any conclusions regarding dietary determinants of B-12 status in this cohort. Dietary B-12 intake was a statistically significant predictor of maternal B-12 biomarker concentrations, even after controlling for prenatal supplement intake in a previous cohort of 386 pregnant women in Canada (156). The use of prenatal supplements has previously been reported to range from 80% to 100% in Canadian cohorts in the 2nd and 3rd trimester, but was only 60% during the 1st trimester of pregnancy (156,181,195,221). Over-the-counter prenatal supplements available on the Canadian market contain between 2.6–12 µg B-12 (284) (RDA: 2.6 µg/d). Visentin et al. (156) reported a 144  significant effect of supplement use on maternal plasma total B-12, MMA, and tHcy concentrations in their Canadian cohort. Since dietary B-12 intake independent of supplement use, and prenatal supplement use, are strong predictors of B-12 status in pregnant women (156), I hypothesize that the lower B-12 biomarker concentrations in women of South Asian ethnicity in this study are due to lower dietary B-12 intake compared to European women. However, Quay et al. (169) found no difference in dietary B-12 intake between reproductive-aged women of South Asian and European ethnicity residing in Metro Vancouver, Canada, despite a tendency for a higher prevalence of B-12 deficiency in women of South Asian ethnicity. Thus, given the high prevalence of pregnant South Asian women classified as B-12 deficient in the present study, it is important to study the impact of dietary B-12 intake and prenatal vitamin supplement use on B-12 status in South Asians in Canada using ethnic-specific, validated tools. Clinical follow-up measures, i.e. hematological or neurological assessment, and data on genetic variants were not available for the women or their newborns in this study. Women and newborns were classified as having ‘impaired functional B-12 status’ based on biomarker, i.e. MMA, concentrations only. Hence, there is the possibility that individuals were misclassified with respect to their B-12 status. As discussed previously, MMA concentrations may be elevated independent of B-status, which might be explained by other confounding factors, such as genetic influences (124). This may affect estimation of the prevalence of deficiency and testing of diagnostic performance. It should, however, be noted that correlations between B-12 biomarker concentration and adverse health outcomes are poor and not always observed (104). B-12 deficiency can exist without the obvious appearance of clinical symptoms. In particular, in newborns and infants, clinical outcomes of inadequate B-12 status 145  are easily missed or misinterpreted, because the symptoms are non-specific (63). No conclusions can be drawn from this study on the physiologic impact of what had been defined as ‘impaired functional B-12 status’ on fetal or infant growth and development or maternal health. Future studies should evaluate the present study’s findings by following infants and mothers over time and using validated age-specific tools to determine infant development and maternal health, respectively. 4.6. Conclusions In summary, this study found a high prevalence of pregnant women classified as B-12 deficient confirming that maternal B-12 deficiency is of concern in Canada. The substantially lower B-12 status observed in pregnant women of South Asian ethnicity warrants further research to identify predictors of B-12 status and allow for targeted interventions. Findings of this study confirmed that the estimation of the prevalence of maternal B-12 deficiency greatly depends on the choice of biomarker and related cutoffs (or reference values). A reference value for serum total B-12 concentrations of <181 pmol/L may provide a more meaningful measure of B-12 deficiency than commonly used non-pregnant adult cutoffs. Pregnancy-specific cutoffs should be established from this reference value to determine B-12 status. Maternal serum total B-12 and holoTC concentrations are equally strong diagnostic tools to identify pregnant women with impaired functional B-12 status. However, maternal serum total B-12, serum holoTC, and serum MMA concentrations during early pregnancy are poor predictors of neonatal functional B-12 status at birth. More research is warranted to 146  identify sensitive maternal biomarker(s) and ideal diagnostic timing for meaningful estimation of fetal B-12 status and stores. 147  5. High Prevalence of Vitamin B-12 Deficiency in Rural Indonesian Infants Followed at 6- to 12-Months of Age 5.1.Summary Prolonged vitamin B-12 (B-12) deficiency in infants has been associated with poor cognitive development. Infants living in low- and middle-income countries (LMIC) may be at increased risk for B-12 deficiency due to low intakes of B-12 from breastmilk and complementary foods. The objectives of the study were to determine the prevalence and predictors of B-12 deficiency in infants living in the Sumedang district of West Java, Indonesia, during the complementary feeding period (ages 6–12 mo). Apparently healthy 6-mo old infants (n= 228), predominantly breastfed from birth to 4 mo, were randomly selected based on local birth registry data and followed at 6, 9 and 12 mo of age. B-12 status was assessed in infants at all time points using serum total B-12 and methylmalonic acid (MMA) concentrations and in mothers at enrollment using dried blood spot (DBS) MMA concentrations. Mixed effects models were used to evaluate the association between infant B-12 status and maternal functional B-12 status. The prevalence of B-12 deficiency (serum total B-12 concentration <191 pmol/L) among infants was 27% and did not differ between study visits. Maternal functional B-12 status, as assessed by DBS MMA concentrations, was a significant predictor of infant MMA concentrations [β (95% CI): -0.00069 (-0.0012; -0.00012); P= 0.004; natural log-transformed], but not total B-12 concentrations. In conclusions, I observed a high prevalence of infantile B-12 deficiency in these Indonesian infants. Given the importance of B-12 for infant development, future research is warranted to determine predictors of this observed high prevalence of infantile B-12 deficiency in rural Indonesia to allow for targeted interventions. 148  5.2.Introduction Prolonged low vitamin B-12 (B-12) status and B-12 deficiency in early life has been related to a range of adverse health outcomes in infants, children, and adolescents, including persistent long-term neurocognitive impairment and anemia (28,33,285). Infantile B-12 deficiency secondary to maternal B-12 deficiency has been associated with brain atrophy and demyelination leading to long-term impaired neurocognitive function (32,33,286). Research conducted in India revealed an association between B-12 status and development and growth in infants and children aged 6–30 mo (74,287,288). Similarly, a study in the Netherlands showed that low intakes of B-12 during infancy are associated with lower cognitive scores in childhood (289). In summary, B-12 adequacy in early life appears to be critical for healthy child development in the long-term. The development of fetal B-12 stores in utero is a critical foundation to achieve adequate B-12 status in the 1st year of life. Offspring of B-12 deficient mothers develop insufficient stores that can be depleted within 1 y if untreated (32). Breastmilk of B-12 deficient women provides insufficient amounts of B-12 to restore infant B-12 stores or improve B-12 status (35,212,213). As exclusive breastfeeding is recommended for the first 6 mo of life, breastfed infants may be at increased risk of B-12 deficiency (219).  Circulating concentrations of the direct and functional biomarkers total B-12 and methylmalonic acid (MMA), respectively, change during the 1st year of life in healthy infants, indicative of a decrease in B-12 status until 6 mo of life followed by an improvement of B-12 status from 6 mo to 1 y of life (116,163); total B-12 concentrations reach a minimum while MMA concentrations reach a maximum at 6 mo of life. Potential reasons include the 149  introduction of B-12 rich complementary foods, gastrointestinal maturation, changes in fetal B-12 stores or other, unknown physiological changes (116,248). Yet, to my knowledge, only 1 longitudinal study (163) has assessed B-12 status of 361 infants at 2 time points, i.e. 6 and 12 mo, in a healthy, breastfed population. Further longitudinal research is needed to fully understand and describe the changes in B-12 status during infancy. Given the current knowledge on changes in B-12 biomarker concentrations during infancy, several studies suggested age-specific reference intervals for B-12 biomarker concentrations, including for infants (0–1 y) (162,163,290,291). The most comprehensive trial investigating pediatric biomarkers in ethnically diverse infants in Toronto, Canada, (CALIPER study) computed a reference value of 259 pg/mL (191 pmol/L) for total B-12 in healthy infants aged 5 d to <1 y (162). The study estimated the 2.5th percentile of serum total B-12 concentrations of 257 healthy infants following current clinical guidelines (CLSI C28-A3) (162,184,292). Given the age-specificity of the reference value, it may provide a more accurate measure of B-12 status in infants than the commonly used adult cutoff (<148 pmol/L). Populations of LMIC may be at increased risk for nutrient deficiencies, including B-12 deficiency (293). Several predictors of B-12 deficiency have been described in LMIC (6). Predictors include low consumption of animal-source foods, fortified foods, or supplements, the only dietary sources of B-12. Low consumption of animal-source foods has been described in Indonesia (205,294). Additionally, overall a high prevalence of micronutrient deficiencies, i.e. vitamin A, zinc, and iron, has been reported in infants living in rural Indonesia [West Java; (223)], yet nothing is known about B-12 status. Given that micronutrient deficiencies in infants commonly co-exist (295) and Indonesian infants may receive complementary foods that do not conform to the World Health Organization guidelines for infant and young child feeding 150  (205,206), I hypothesize that infants living in rural Indonesia are at increased risk for B-12 deficiency. In light of the crucial role of B-12 for growth and development, the primary objective of this study was to determine the prevalence of B-12 deficiency during the introduction of complementary foods among infants living in the Sumedang district of West Java, a rural setting in Indonesia. I also explored the changes in total B-12 and MMA concentrations from 6 to 12 mo of age as well as the association of infant B-12 status with maternal B-12 status. 5.3.Methods 5.3.1. Participants and Study Design This is a secondary analysis of a prospective, longitudinal study designed to evaluate the nutritional status and growth of apparently healthy, breastfed infants aged 6–12 mo in Indonesia. The study was conducted between August 2013 and August 2014 in 30 villages of the Sumedang district of West Java, a low-income, rural setting in Indonesia. Infants aged 6 mo were randomly selected based on local birth registry data. Only infants, exclusively/predominantly breastfed up to 4 mo of age, apparently healthy, without severe anemia (hemoglobin <7 g/dL) or severe acute malnutrition (weight-for-height z-score <-3), were enrolled in the study. A sample size of 200 healthy, breastfed infants was calculated based on the prevalence of stunting, with 228 infants (83.8% of eligible infants selected) enrolling in the study at 6 mo of age. Of these infants, 202 and 190 attended study visits at age 9 and 12 mo of age, respectively.  151  At enrollment, sociodemographic and health questionnaires were collected together with anthropometric measurements and blood sampling of mother and infant. Infant blood samples were collected again at 9 and 12 mo of infant age.  The present study was conducted according to the guidelines in the Declaration of Helsinki and ethical approval of the study protocol was obtained from the University of Otago (H14/022), Universitas Padjadjaran (No 132/UN6C2.1.2/KEPK/PN/2014) and the Clinical Research Ethics Board at The University of British Columbia (H15-00106). Written informed consent was obtained from the parents or primary caregiver of the infant before enrollment. 5.3.2. Questionnaires and Anthropometric Measures Pre-tested questionnaires on socioeconomic and health status were administered to the mother or primary caregiver by trained health workers in the participants’ homes. Socioeconomic status was measured using an asset index, created by combining data on household possessions and characteristics using principal component analysis. The index was divided into tertiles for use as covariate in the analyses, and is referred to as household wealth score. All anthropometric measurements, including weight and length of nude infants and height and weight of lightly clothed mothers were taken in triplicate using standardized techniques. 5.3.3. Blood Collection Infant blood samples were collected by venipuncture into trace-element free monovette tubes (Becton Dickinson) at all 3 study time points. Sample tubes were immediately stored 152  inside a cool box. Within 2 hours after collection, serum was separated, aliquoted and stored at -20 ºC.  Maternal dried blood spot (DBS) samples were collected at enrollment only. Either a middle or index finger was pricked after disinfecting with an alcohol swab. The first drop of blood was discarded to avoid any contamination of the sample with tissue cells. Blood was allowed to create a circle (11-mm diameter) on a filter paper (Whatman 903). After collection, samples were dried at room temperature for 24 h before being transferred into sealable biohazard bags containing a desiccant and placed at -20ºC.  All frozen samples were transported on dry ice to the University of Otago, Dunedin, New Zealand, for infant serum B-12 analyses. The remaining infant serum aliquots and maternal DBSs were shipped on dry ice to The University of British Columbia, Vancouver, Canada, for MMA quantification. 5.3.4. Biomarker Quantification and Definition of Vitamin B-12 Status  Serum total B-12 concentrations were quantified using Elecsys® 2010 (Roche Diagnostics) automated electrochemiluminescence immunoassay. A reference value of <191 pmol/L (259 pg/mL) for infants aged 0–12 mo has been suggested based on a reference interval (2.5th percentile) of 257 ethnically diverse, partially breastfed, healthy infants of mostly B-12 supplemented mothers residing in Toronto, Canada (162,292). Circulating total B-12 concentrations <220 pmol/L have been associated with elevated MMA concentrations reflecting intracellular B-12 deficiency in all age groups (21). 153  Serum and DBS MMA concentrations were determined as previously described (Chapter 2.3). The inter-assay CVs were 2.2% (n= 5) for serum and 5.0% (n= 7) for DBS analyses based on pooled samples. For consistency, DBS concentrations will be presented as pmol MMA per 8-mm punch (pmol/punch). A reference value, suggestive of functional B-12 deficiency, of 5.03 pmol/punch was computed as 97.5th percentile of DBS MMA from a subset of healthy women (19–35 y; n= 146), enrolled in a cross-sectional study in Vancouver, Canada (169), who had serum MMA concentrations <260 nmol/L (data not shown). A large decrease in circulating MMA concentrations have been suggested in infants from the age of 6 mo to 12 mo (116,163). No age-specific cutoffs or reference values for infant serum MMA concentrations have yet been established. Thus, only serum total B-12 concentration was used for classifying the infants into adequate (total B-12 ≥220 pmol/L), suboptimal and deficient (total B-12 <191 pmol/L) B-12 status. Inadequate B-12 status refers to combined suboptimal and deficient B-12 status (total B-12 <220 pmol/L). Serum MMA concentration was included in the models to identify predictors of B-12 status. 5.3.5. Statistical Analyses Statistical analyses were performed using Stata/IC (Stata Corp) version 14.2 for Windows 10. Normality of distribution of the continuous variables was tested using Shapiro-Wilk test (236). Unless stated otherwise, data are presented as geometric mean with 95% confidence interval (CI). Predictor variables were determined using linear mixed effects models accounting for repeated measures. Residuals and random intercepts of infant serum total B-12 and MMA concentrations were not normally distributed; modelling was performed on natural log-154  transformed data. P-values presented for mixed effects models are based on likelihood-ratio tests. Significant differences within biomarker concentrations and prevalence of B-12 deficiency across the 3 study time points were determined after pairwise comparison with Bonferroni adjustment as post-estimation for the mixed effects models. Changes of infant B-12 biomarker concentrations with age were modelled using infant age as a continuous variable, not the study time point. Maternal DBS MMA concentrations as a predictor of infant total B-12 or MMA concentrations were determined after adjusting for infant age, sex, maternal age, education, and household wealth score (in tertiles). A pseudo-R2 was estimated for the mixed effects models by correlating the observed outcome variable with the predicted outcome variable and squaring the correlation. However, this does not account for repeated measures. A P-value of <0.05 indicated statistical significance in all analyses. 5.4.Results 5.4.1. Participant Characteristics The final cohort for analysis was comprised of 228 mother-infant dyads. At enrollment, 16% of infants were classified as stunted (length-for-age z-score <-2); and 2% were classified as wasted (weight-for-length z-score <-2). The prevalence of stunting at 9 and 12 mo of infant age increased to 19% and 23%, respectively, whereas wasting remained relatively stable at 2% and 3%, respectively.  Ninety-nine percent of infants were breastfed at enrollment with the majority of infants continuing to be breastfed until 12 mo of age (>97%). The mean (95% CI) age of the mothers was 26.7 (25.7; 27.5) y. More than one-half of mothers (54%) achieved a secondary school education, 40% had a primary school education or less and the remaining 155  women (6%) had attended college or university. The majority of participants were classified in the middle household wealth tertile (n=138; 61%). 5.4.2. Infant and Maternal Vitamin B-12 Status The prevalence of B-12 deficiency [serum total B-12 <191 pmol/L (162)] and inadequate B-12 status (serum total B-12 <220 pmol/L) in infants did not differ between 6-, 9- and 12-mo of age and was ~27% and ~38%, respectively, across all time points (Figure 5-1). MMA could be quantified in 184 DBSs of mothers. Twenty-three percent of mothers (n= 42) had elevated DBS MMA concentrations, suggestive of functional B-12 deficiency, at enrollment. 156   Figure 5-1: Prevalence (95% CI; Wald estimation) of infants [grey; n: 165 (enrollment), 159 (3-month follow-up), 143 (6-month follow-up)] with vitamin B-12 (B-12) deficiency [serum total B-12 <191 pmol/L (162); dark grey] and inadequate B-12 status (serum total B-12 <220 pmol/L; light grey), and mothers (black; n: 184) with dried blood spot methylmalonic acid concentrations suggestive of functional B-12 deficiency.  Mean (95% CI) infant serum total B-12 concentration did not change significantly over time (Table 5-1) and was 250 (239; 262) pmol/L across all time points. Serum MMA concentration was lower at 9-mo and 12-mo compared to 6-mo of age (P= 0.001).  157  Table 5-1: Vitamin B-12 (B-12) status [geometric mean (95% CI)] at 3 study visits and change over time in infants living in the Sumedang District of West Java in rural Indonesia.1   6-month 9-month 12-month P-value2 Infant age (mo) 6.62 (6.58; 6.67) n= 228 9.74 (9.67; 9.81) n= 201 12.9 (12.8; 13.0) n= 190 N/A B-12 biomarker      Total B-12 (pmol/L) 242 (223; 262) n= 165 264 (242; 288) n= 159 243 (225; 263) n= 143 0.08  MMA (nmol/L) 571 (509; 642)a n= 117 457 (411; 509)b n= 119 504 (446; 569)b n= 116 0.001 1 MMA: methylmalonic acid; Superscript letters indicate significant difference (P< 0.05) within a row, i.e. change between study visits. 2 Likelihood ratio test after mixed effects models adjusted for repeated measures using infant serum total B-12 or MMA concentration (natural log-transformed) as dependent variable and study visit (categorical) as independent variable.   5.4.3. Predictors of Infantile Vitamin B-12 Status Neither total B-12 nor MMA concentration (natural log-transformed) was significantly predicted by infant age (Figure 5-2). In the linear mixed effects model, infant serum total B-12 concentration was a significant predictor of infant serum MMA concentration (natural log-transformed) across all study time points (Figure 5-3).  158   Figure 5-2: Serum total vitamin B-12 (B-12; n= 146) and methylmalonic acid (MMA; n= 115) concentrations by infant age in months (mo). P-value determined by likelihood-ratio test after mixed effects model adjusted for repeated measures using infant serum total B-12 or MMA concentrations (natural log-transformed) as dependent variable and infant age (continuous) as independent variable.    159   Figure 5-3: Serum methylmalonic acid (MMA; natural log-transformed) concentration (nmol/L) by serum total vitamin B-12 (B-12) concentration (pmol/L) in infants aged 6 (square; n= 117), 9 (circle; n= 122), and 12 (cross; n= 116) months.  Red line depicts linear relationship as determined by mixed effects model adjusted for repeated measured with infant serum MMA concentration (natural log-transformed) as dependent and infant serum total B-12 concentration as independent variable.  Maternal DBS MMA concentration at enrollment was a significant predictor of infant serum MMA concentration (natural log-transformed; nmol/L) in the linear mixed effects model after adjusting for infant age, sex, maternal age, education, and household wealth score as well as repeated measure (Figure 5-4); a pseudo-R2 for the entire model of 0.18 was estimated. However, maternal DBS MMA concentration at enrollment was not a significant predictor of infant serum total B-12 concentration (P= 0.4). 160   Figure 5-4: Infant serum methylmalonic acid (MMA; natural log-transformed) concentration (nmol/L) by maternal dried blood spot (DBS) MMA concentration (pmol/8-mm DBS) in infants aged 6 (square), 9 (circle), and 12 (cross) months and their mothers at 6 months postpartum.  Red line depicts linear relationship as determined by mixed effects model adjusted for repeated measures, infant age, sex, maternal age, education, and household wealth score, with infant serum MMA concentration (natural log-transformed) as dependent and maternal DBS MMA concentration as independent variable.  5.5.Discussion Vitamin B-12 is crucial for infant growth and development. To my knowledge, this is the first study to report on the prevalence of B-12 deficiency in Indonesia. In the present study, about 27% of exclusively/predominantly breastfed (up to 4 mo of age) infants, living in rural Indonesia, were classified as B-12 deficient using an age-specific reference value [serum total B-12 <191 pmol/L (162)]. Thirty-eight percent of infants had serum B-12 concentrations 161  <220 pmol/L, indicating inadequate B-12 status. No significant changes in infant serum total B-12 concentration or prevalence of deficiency were observed across the study period. The prevalence of maternal functional B-12 deficiency (as assessed by elevated DBS MMA) at 6 mo postpartum was similar to that of infant B-12 deficiency (23% and 29%, respectively). The findings of this study suggest that maternal and infant B-12 deficiency are prevalent in rural Indonesia. Two non-representative studies have suggested infant-specific reference intervals for circulating total B-12 concentrations with lower limits of 124 pmol/L (6 mo) and 197 pmol/L (12 mo), and 88 pmol/L (4–37 wk), respectively (163,290). Both studies (n: 361 and 509) included only healthy infants, mostly of European mothers residing in Norway and Australia, respectively, from households of a diverse socio-economic status (163,290). Some infants were being breastfed (84% at 6 mo and 47% at 12 mo, and 60%, respectively), and some infants received formula (16% at 6 mo and 31% at 12 mo, and 14%, respectively) (163,290). As such, both reference intervals were derived from a diverse sample of infants of, yet, mostly European ethnicity. A significant difference in serum total B-12 reference values for breastfed and non-breastfed infants has been described and related to higher dietary intakes of B-12 in non-breastfed infants from formula (163). The prevalence of infants receiving formula in the present study was low (~5%; data not shown). Breastfeeding frequency likely varied in the infants across time, since the infants were being introduced to complementary foods. Recently, a reference value of <191 pmol/L (259 pg/mL) for all infants aged 0–12 mo has been suggested, independent of breastfeeding status, based on a reference interval (2.5th percentile) of 257 ethnically diverse, partially breastfed, healthy infants residing in Toronto, Canada (CALIPER study) according to current clinical guidelines, (162,292). The use of a recent 162  reference value for B-12 deficiency determined on partially breastfed infants of diverse ethnicity seemed an appropriate measure in this study population. However, it has to be noted that the reference value was derived from a population in Canada where prevalence of maternal supplement intake during pregnancy was likely high. Prevalence of supplement use was not reported in the CALIPER study (292), however, prenatal supplement use among pregnant women in Canada has been reported to be high (80–100%) (81,156,221). Maternal supplement use has been associated with a higher B-12 status in the offspring (156) and, thus, the Canadian reference value might not truly represent an appropriate reference sample (184). Yet, most prenatal supplements in Canada contain B-12 amounts at the recommended dietary allowance (RDA; 2.6 µg) (284) leading to a dietary B-12 intake of approximately twice the RDA or higher in Canadian women (195). In summary, the use of an age-specific reference value allowed for a more appropriate assessment of B-12 status in these infants until reliable cutoffs are established (162). Further, while the CALIPER study used the Abbott ARCHITECT i2000 to quantify serum total B-12 concentrations (162), the present study used the Roche Elecsys, as automated analyzer to quantify serum total B-12 concentrations. Differences in instrument performance can influence cutoffs and as such prevalence of deficiency determined in the present study. Overall, a good agreement between both instruments of r= 0.95–0.99 (Deming regression) has been described (90,94). However, Vogeser et al. (94) suggested an adjustment equation to account for systematic bias. Using this equation, an adjusted reference value of 221.5 pmol/L can be computed from the CALIPER reference value of 191 pmol/L. As such, the results from the present study may be underestimating the prevalence of B-12 deficiency in rural Indonesian 163  infants. The reference value used to determine B-12 inadequacy (<220 pmol/L) may more accurately reflect deficiency. To date, a limited number of studies have investigated B-12 status among infants worldwide. The prevalence of inadequate B-12 status reported for infants in LMIC are similar to these findings, including 40% in infants aged between 2 and 12 mo in Nepal (serum total B-12 <200 pmol/L), 39% in infants aged 12 mo in Guatemala City, Guatemala, and 43% in infants aged 3 mo in Dhaka, Bangladesh (both: serum total B-12 <220 pmol/L) (213,295). In comparison, a lower prevalence of B-12 deficiency has been found in Norwegian infants aged 12 mo, of whom 5% had serum total B-12 concentrations <197 pmol/L and Canadian infants aged between 5 d and 12 mo, of whom 2.5% had serum total B-12 concentrations <191 pmol/L (162,163). In conclusion, the present results confirm findings that B-12 deficiency and B-12 inadequacy are prevalent and are of concern in infants living in LMIC.  One direct and 1 functional biomarker — serum total B-12 and MMA (130), respectively — were used to determine infant B-12 status in this study. MMA has been described as a sensitive and specific functional indicator of B-12 status in a variety of populations, including infants (21,22,114). Maternal functional B-12 status (as assessed by DBS MMA) at 6 mo postpartum weakly but statistically significantly predicted infant MMA concentrations over 6–12 mo of age. On the other hand, maternal MMA concentration was not associated with infant serum total B-12 concentration. Correlations between maternal and infant B-12 status have previously been discussed. Two studies in Guatemala reported a significant positive association between maternal and infant serum total B-12 concentrations at 12 mo postpartum (219,220). To my knowledge, this is the first study comparing predictors of direct and functional infant B-12 biomarkers. It is not understood whether the observed 164  association between maternal and infant MMA concentrations, only, in the present study is due to maternal B-12 status during pregnancy determining fetal and infant B-12 stores (220) and postpartum maternal B-12 status or due to other unknown reasons. Some genetic predictors of MMA concentrations, independent of B-12 status, have recently been described and may contribute to the association of maternal and infant MMA concentrations (124).  Changes in B-12 biomarker concentrations over time in healthy infants have been previously discussed to understand infant B-12 requirements (116). It has been described that circulating MMA concentration decreases substantially, while circulating total B-12 concentration increases from age 6 to 12 mo (116,163). However, results from this study do not confirm these findings. Neither serum total B-12 nor serum MMA concentration was significantly predicted by infant age, which may be due to sample size. However, mean serum MMA concentration decreased significantly from 6-mo to 9-mo and 12-mo of infant age. Thus, there was a trend that MMA concentration may decrease weakly while total B12 concentration remains unchanged. More research is needed to fully understand the changes in infant B-12 biomarker concentrations and related predictors and determinants. The present study has several strengths, including the measurement of infant B-12 status at multiple time points by multiple biomarkers, and the use of an age-specific reference value. However, there are some limitations to the study which require future research. First, the present study does not reveal any information on the predictors of the observed high prevalence of B-12 deficiency in infants living in rural Indonesia. Complementary foods low in animal-source foods as well as maternal B-12 deficiency and breastfeeding frequency have been discussed as determinants of infant B-12 deficiency in LMIC (219). Gastro-intestinal disease in the infant may also contribute to B-12 deficiency (6). Second, in the present study 165  maternal B-12 status was assessed by DBS MMA only. Although I have shown a strong correlation between plasma and DBS MMA concentrations in healthy, young women (Figure 2-4), MMA quantified in plasma or serum with an additional measurement of a direct B-12 biomarker, i.e. total B-12 or holoTC, is the recommended method for assessing B-12 status (126). Thus, future research is needed to identify predictors of infant B-12 deficiency in rural Indonesia, including comprehensive measures of maternal B-12 status. 5.6.Conclusions In conclusion, the present study found a high prevalence of B-12 deficiency and inadequate B-12 status in healthy infants aged 6–12 mo and their mothers at 6 mo postpartum, living in rural Indonesia. Maternal functional B-12 status appeared to be weakly related to infant functional B-12 status. Given the importance of B-12 for infant development, further research on predictors of infant B-12 status allowing for interventions improving B-12 status in infants in rural Indonesia is warranted. 166  6. General Discussion, Conclusions and Future Directions 6.1.Summary of Presented Work Vitamin B-12 (B-12) is involved in biological processes crucial during stages of rapid growth (Chapter 1). My PhD work focused on the development, evaluation, and application of screening tools for B-12 deficiency during fetal and infant development. Overall, this research has contributed to the knowledge on assessment and prevalence of perinatal, neonatal, and infantile B-12 deficiency. It is the first to introduce dried blood spots (DBSs) as a novel means to assess B-12 status in newborns and populations in remote settings. I report on the prevalence of B-12 deficiency in pregnant women of South Asian and European ethnicity residing in Metro Vancouver, Canada, and their newborns; and I am the first to describe the prevalence of B-12 deficiency in infants transitioning to complementary foods in rural Indonesia and their mothers. In summary, I developed a novel means to assess B-12 status and applied it in studies of vulnerable populations, such as a pregnancy cohort in Vancouver, Canada, and a study in infants and their mothers in rural Indonesia. This chapter presents a brief summary of all research outcomes, followed by an overall discussion of the findings in the larger context of B-12 research. The major limitations of the studies are discussed. The chapter concludes with suggestions for future research. In Chapter 2, I described a precise assay to quantify methylmalonic acid (MMA) in DBSs of healthy adults using stable isotope dilution liquid chromatography-tandem mass spectrometry [LC-MS/MS; recovery: 93-98%; inter-assay coefficient of variation (CV): <7%; intra-assay CV: <2%]. In a sample of healthy young adult women, DBS and serum MMA concentrations were significantly correlated (r= 0.97; P< 0.001). Serum MMA concentration 167  has so far been the gold standard for functional B-12 status assessment (8,114). Additionally, I showed that DBS MMA concentration remains stable for at least 8 weeks (wk) when DBSs are stored at refrigerator temperature (2–4°C) or for at least 1year (y) when stored frozen (-80°C). Thus, DBS MMA has the potential to be a convenient tool to screen for functional B-12 status. In Chapter 3, I calculated a reference interval of DBS MMA concentrations in healthy, term newborns of 9.89–29.3 pmol/8-mm punch (0.45–1.33 µmol/L whole blood). DBS MMA concentrations of patients with methylmalonic acidemia were above the upper limit of the reference interval demonstrating specificity of the interval. The storage of DBSs at room temperature during routine handling of newborn screening cards for 2 wk had no significant effect on the reference interval. These findings suggest that newborns with MMA concentrations >29.3 pmol/8-mm punch in routinely collected DBSs display ‘abnormal’ circulating MMA concentrations that may be indicative of functional B-12 deficiency. In Chapter 4, I showed that pregnant women of South Asian ethnicity residing in Metro Vancouver had a significantly lower B-12 status than Europeans, e.g. during the first trimester, mean (95% CI) serum total B-12 concentrations were significantly lower in South Asian compared to European women [189 (180; 199) pmol/L versus 246 (236; 257) pmol/L]. Pregnant women of South Asian compared to European ethnicity also showed a significantly lower B-12 status when measuring serum MMA and holoTC concentrations. This difference in B-12 status was not reflected in the DBS MMA concentrations of their newborns. In summary, pregnant women of South Asian compared to European ethnicity have a substantially lower B-12 status which has in previous research been associated with adverse pregnancy outcomes, including low birth weight and neural tube defects (NTDs). 168  I evaluated the utility of total B-12 and holoTC as direct indicators of B-12 status in pregnant women, as described in Chapter 4. Serum total B-12 and holoTC performed equally well [area under the curve (AUC): ~0.8] in diagnosing pregnant women with elevated serum MMA concentrations (>210 nmol/L). However, the capacity of any of the maternal biomarkers, i.e., total B-12, holoTC, or MMA, assessed during early pregnancy (1st and 2nd trimester) to predict neonatal elevated MMA concentration (DBS MMA >75th percentile) was low (<5%). I derived a new, pregnancy-specific reference value for diagnosing maternal B-12 deficiency of total B-12 <181 pmol/L using cut-point analysis. The use of pregnancy-specific reference values may allow for a more accurate assessment of B-12 status during pregnancy. Lastly, I found that the prevalence of B-12 deficiency is high (27%) among infants aged 6–12 months (mo), and their mothers (23%), living in rural Indonesia (Chapter 5). Infant functional B-12 status was predicted by maternal functional B-12 status (assessed by DBS MMA) [β (95% CI): -0.00069 (-0.0012; -0.00012); P= 0.004; natural log-transformed]. The findings suggest that future research is needed focusing on improving B-12 status of infants and their mothers in rural Indonesia. In conclusion, my findings suggest that DBS MMA is a convenient screening tool for B-12 status and may be used in at-risk populations, including newborns. Using DBS MMA, I found in a pregnancy cohort that maternal B-12 status during early pregnancy is only weakly related to neonatal functional B-12 status; and in a study in rural Indonesia that maternal and infant functional B-12 status are weakly, but statistically significantly, related during the introduction of complementary foods. Further, my research confirmed the hypotheses that B-12 deficiency is prevalent in South Asian minorities in British Columbia, Canada, and vulnerable population groups living in Indonesia, a low- and middle-income country (LMIC).  169  6.2.General Discussion of the Key Findings 6.2.1. Dried Blood Spots as a Novel Mean to Assess Vitamin B-12 Status Dried blood spot collection has been described as a convenient, minimally-invasive, and cost-effective means to obtaining biological samples (138,139). Collection of DBSs requires a much lower blood volume and less logistic effort compared with venipuncture (142). The use of DBSs allows maximizing participation and collection of biological samples within existing public health programs, i.e., newborn screening programs, and from populations in remote settings (296,297). In my research, I showed that the collection and analysis of DBSs from the BC Newborn Screening Program and from lactating women in rural Indonesia was feasible; thus, minimally invasive B-12 status assessment in these vulnerable population groups is possible. This allowed for B-12 status assessment that, compared with the use of dietary data, is not confounded by self-report bias (141). Methylmalonic acid, the most specific functional indicator of B-12 status (8,114), was chosen as B-12 biomarker to be quantified in DBSs. MMA in biological samples is stable at room temperature for at least 1 wk and sensitive quantification methods are available using LC-MS/MS (85). As such, MMA has ideal biological and analytical properties to serve as target B-12 biomarker in DBSs. Circulating MMA concentration, however, is affected by renal function and other confounders, such as age (8). Renal function increases during healthy pregnancy and infancy due to pregnancy-related physiological changes and infant organ maturation, respectively (118,248). The altered renal function can impact circulating biomarker concentrations; accordingly pregnancy- and age-specific reference intervals are needed. I have presented age-170  specific reference intervals, computed according to clinical standards (184), for DBS MMA concentrations in newborns (Chapter 3) and young-adult women (Chapter 5.3.4). When specific reference intervals are not available (e.g., pregnant women), or for population groups prone to renal malfunction (e.g., older adults) (115), MMA concentrations should be evaluated in combination with markers of renal function. Renal clearance is commonly assessed by measuring creatinine concentrations in plasma or urine (84). There are no methods available to determine creatinine concentrations in DBSs (139), only in dried urine spots (239). The use of dried urine spots, however, requires additional sample collection, a separate extraction method, and the set-up of a second LC-MS/MS method. MCA, in combination with MMA, has been suggested to be a valid biomarker for renal function (119). As discussed in Chapter 1.2.2, using the MCA to MMA ratio showed high sensitivity and specificity in diagnosing older adults with elevated MMA concentrations due to impaired renal function (defined as serum creatinine >1.7 mg/dL) (119). Methods for DBS MCA quantification are available (122,135). However, DBS MCA was not quantified in the present studies of newborns and reproductive-aged women, because these study populations are only at minimal risk of renal abnormalities. In summary, assessment of functional B-12 status using DBS MMA is, to date, possible in newborns and young-adult women using the reference intervals presented in this work. Simultaneous quantification of MMA and MCA in DBSs and the calculation of additional population-specific reference intervals might allow for functional B-12 status assessment in the older adults and pregnant women, respectively, using the convenient DBS method. Propionate-producing gut microbiota have been hypothesized to affect circulating MMA concentrations. It has been suggested that gut microbiota can produce >20% of total circulating propionate (298), which then can be catabolized into MMA (Figure 1-1). As such, 171  an altered or immature gut microbiota may lead to differences in circulating MMA concentrations. This may affect DBS MMA concentrations of newborns with altered gut microbiota. Factors such as gestational age, mode of delivery, environment, hygienic measures, and diet have been suggested to influence the gut microbiota in the newborn (299). Additionally, gut microbiota may differ between ethnic groups and in individuals taking antibiotics (300). At this point, however, the association between the gut microbiome and circulating MMA concentrations are not fully understood and research in populations, especially newborns, with altered gut microbiota are needed to further elucidate on the validity of the reference interval for DBS MMA concentration in newborns. Hematocrit can affect the analyte concentration in a DBS by affecting the viscosity of blood and, thus, the blood volume per spot (141,297). Blood volume per spot is generally estimated to be ~3.2 µL/3-mm punch (typical punch size in newborn screening) with little variation for newborns (301). As such, the reference interval presented for neonatal DBS MMA is valid without controlling for hematocrit. However, hematocrit in adults can vary substantially and, hence, can affect the concentration of MMA in DBSs (141,142). Quantification of hemoglobin or other measures of hematocrit in DBSs may allow controlling for the hematocrit effect. Yet, I found a strong correlation (r= 0.97; P< 0.001) between plasma and DBS MMA concentrations in healthy, young adult women, implying that DBS MMA may be a valid proxy of assessing plasma MMA concentrations, the ‘gold standard’ of functional B-12 status assessment. Nevertheless, more research is needed to determine the effect of hematocrit on DBS MMA concentrations in adults, especially given that B-12 deficiency can lead to anemia, which affects the hematocrit and lowers the viscosity of blood. Thus, DBS 172  MMA concentrations of anemic individuals may be falsely low, leading to falsely negative results. Cutoffs for a biomarker are quantitative measures for a health condition in a population and are validated by comparing them to the gold standard of practice (12). No validated cutoffs for DBS MMA concentration for B-12 status assessment have yet been established from the reference intervals. I estimated from reference intervals of healthy populations that DBS MMA concentrations in newborns >29.3 pmol/8-mm punch and in young adult women >5.08 pmol/8-mm punch identify individuals with ‘abnormal’ MMA concentrations (184), potentially reflecting intracellular B-12 deficiency. Estimating reference intervals, e.g. 97.5th percentile of a reference population, is standard clinical and common practice to classify individuals (184). However, individuals outside the reference interval may not necessarily present with the health condition (302), i.e. in this case B-12 deficiency. Thus, further research is needed to evaluate and validate the reference intervals (12). The diagnostic utility, e.g. receiver operating characteristics (ROC) analysis, of the reference intervals to identify newborns or women with adverse health outcomes related to B-12 deficiency should be determined. Additionally, further research is needed that includes clinical follow up of newborns who were diagnosed as B-12 deficient using the DBS MMA reference interval to confirm B-12 deficiency either through measuring an additional biomarker of B-12 status (e.g. serum total B-12), dietary assessment or blood testing of their mothers, or genetic testing. This would allow generating more clinically meaningful and validated cutoffs. Until then, the proposed reference intervals should be interpreted with caution, and additional follow-up is recommended at this point to confirm B-12 deficiency. 173  Whatman 903 protein saver cards (GE Healthcare) are the standard tool for DBS sample collection. The cards are manufactured in accordance with ISO9001, with strict quality control to ensure homogenous composition as well as uniformity in thickness, flow rate, absorbancy, and purity. They are designed to lyse red blood cells and stabilize metabolites. However, I found that DBS MMA concentrations increase significantly over time, unless stored frozen; DBS cards used for MMA analysis should not be left at room temperature (18–22 °C) for >1 wk. Nevertheless, the upper limit of the neonatal DBS MMA reference interval was not affected when DBSs were left at room temperature for 2 wk. DBS MMA concentrations are stable at refrigerator temperature (2–4°C) for >8 wk or at -80°C for at least 1 yr. In summary, DBS MMA is a convenient tool to estimate functional B-12 status in a variety of populations. It allows for minimally invasive screening of populations, for whom other biological measures may not be feasible. However, quantification of 1 direct biomarker in plasma or serum in addition to MMA (126) should be conducted to confirm B-12 deficiency while controlling for confounding factors until valid cutoffs have been established. 6.2.2. Screening of Vitamin B-12 Status Using Biomarkers Valid biomarkers have the advantage of providing valuable insights into a population’s prevalence of nutritional inadequacy without self-report bias or missing cases of non-dietary nutritional deficiencies, such as the ones caused by gastro-intestinal diseases or inborn errors of metabolism (8). Four biomarkers are available to assess B-12 status (114). However, no consensus has yet been reached regarding the most valid B-12 biomarker in pregnant women or infants. In this study of pregnant women (Chapter 4), I hypothesized that maternal serum 174  holoTC concentration during early pregnancy is more strongly correlated with neonatal B-12 status compared to maternal serum total B-12 concentration, and that holoTC is a more meaningful indicator of B-12 status during early pregnancy. This hypothesis was based on the physiological importance of holoTC, i.e., being the B-12 form readily available to the fetus (88,143). However, maternal holoTC was only a marginally stronger predictor of neonatal functional B-12 status (as assessed by DBS MMA) compared to maternal total B-12. Both biomarkers appeared to provide little insight into whether a mother’s offspring will be B-12 deficient at birth. This may, in part, be explained by the preferential unidirectional transport of B-12 to the fetus throughout pregnancy protecting the fetus from developing B-12 deficiency in utero (Chapter 4.5.2). Thus, measuring maternal B-12 status during early pregnancy, regardless of the biomarker used, may have little utility in predicting neonatal B-12 status. However, either biomarker had a strong capacity in diagnosing pregnant women with elevated MMA concentrations (>210 nmol/L), indicative of impaired intracellular B-12 function. Findings from this study suggest that screening for maternal B-12 status during early pregnancy, using holoTC or total B-12, can provide insight into the mothers B-12 status but only poorly predict newborn’s B-12 status. Assessment of perinatal B-12 deficiency using biomarkers, however, is limited by the lack of validated, pregnancy-specific cutoffs. The substantial decrease in total B-12 concentration throughout pregnancy has been discussed in Chapter 1.2.2 (Figure 1-2). Additionally, I reported a significant mean decrease in serum total B-12 concentrations of 3.5 pmol/wk between 8 and 21 weeks of gestation in what is (to date) the largest cohort study of pregnant women (n=751). The decrease may be partially attributed to hemodilution (149) and may not truly reflect B-12 deficiency (87,151). The finding that serum MMA concentration 175  remained largely unchanged would support this hypothesis, indicating that there was no decrease in intracellular B-12 function despite the decrease in total B-12 concentration. The relative decrease per week was similar for serum total B-12 and serum holoTC concentration, suggesting that total B-12 concentration decreases at the same rate as the active form of B-12 that is available to the tissues. The substantial decrease in both biomarkers might lead to a false-positive diagnosis of B-12 deficiency during pregnancy when using non-pregnant adult cutoffs or reference values. The reference value that I computed in this study of 751 Canadian women for total B-12, i.e., 181 pmol/L, may provide a more accurate measure of B-12 status in pregnant women. However, classification of pregnant women using this reference value should be interpreted with caution until the reference values have been validated against clinical outcomes of perinatal B-12 deficiency. Further, assessment of an additional functional biomarker of B-12 status, e.g. MMA, should be considered to confirm B-12 status (126). In the study of rural Indonesian infants (Chapter 5), 1 direct and 1 functional biomarker of B-12 status were quantified as is recommended for B-12 status assessment (126). The lack of established and validated age-specific cutoffs makes B-12 status assessment challenging in this population (8). A reference interval for infant total B-12 concentration has recently been suggested (162). Using this reference interval, the prevalence of B-12 deficiency was 27% in 6–12 mo old infants in rural Indonesia. MMA concentrations have previously been reported to decrease substantially from 6 to 12 mo of life (116,163). A similar tendency was found in the present study. Mean MMA concentrations decreased significantly from 6- to 9-months of age, however, age was not a significant predictor of infant MMA concentrations. No specific reference interval for infants has yet been computed for MMA concentration. Thus, infant MMA concentrations were not used to determine B-12 deficiency in the present study. 176  Using multivariate linear regression, I found infant MMA concentration, not total B-12 concentration, was weakly, but statistically significantly, predicted by maternal MMA concentration. In summary, a recent reference value allowed estimating the prevalence of B-12 deficiency using serum total B-12 concentrations in infants living in rural Indonesia; however, more research is needed to fully elucidate the determinants of B-12 status in this vulnerable population.  In summary, this research contributed to the body of knowledge that biomarkers can provide insight into the B-12 status, especially of pregnant women and also infants. HoloTC and total B-12 were shown to be strong diagnostic tools for B-12 deficiency in pregnant women. A new computed reference value for total B-12 will allow for more accurate B-12 status assessment in pregnant women. However, the reference value should be evaluated and a validated cutoff established.  Self-reported data on dietary intake, on the other hand, can provide information about a populations’ dietary patterns, nutrient intakes, and also nutrient status (303). Knowledge on food intake and behavior is necessary to inform dietary guidance and policies (303). Dietary assessment has been shown to allow for assessment of the nutritional B-12 status of pregnant women (54,156,187,221) and infants (219,304). Dietary B-12 intake, assessed by food frequency questionnaires, was associated with B-12 status, independent of supplement use, in pregnant women with mean dietary B-12 intake ranging from ~3–6 µg/d [estimated average requirement (EAR): 2.2 µg/d] (54,156,187).  Food frequency questionnaires, however, have been reported to overestimate dietary B-12 intake in pregnant women, infants, and children (0–19 y) compared to 24-h recalls, estimated dietary records, or weighed dietary records by 10%–160% as discussed in 2 systematic reviews (305,306). In addition to being limited by 177  common over- or under-reporting and missing non-diet related cases of B-12 deficiency, dietary assessment is prone to a higher variability compared with the use of biomarkers (303). Further, 1 recent study has suggested that the current RDAs for B-12 for pregnant women are set too low, based on evidence that after B-12 consumption even at 3-times the RDA, circulating B-12 was partitioned towards the bioactive form, i.e., holoTC, not the inactive form in pregnant women (307). Taken together, dietary assessment alone, especially when using food frequency questionnaires, may overestimate B-12 status in pregnant women and infants while resulting in a falsely low prevalence of inadequate B-12 intake. As such, biomarker assessment should be the preferred tool to determine B-12 status in a population and individuals, and efforts should focus on establishing validated cutoffs to determine B-12 deficiency related to adverse health outcomes in pregnant women, newborns and infants. 6.2.3. Populations with a High Prevalence of Vitamin B-12 Deficiency Low dietary B-12 intake and impaired B-12 absorption are leading causes for developing B-12 deficiency (6). Low intake of animal-source foods or fortified replicas, the most important dietary sources of B-12, and low vitamin supplement use are predictors of B-12 deficiency world-wide (6). Identifying population groups with a high prevalence of micronutrient inadequacies is important to allow for targeted interventions and prevention of micronutrient deficiencies. In my research, I found that in Metro Vancouver, Canada, pregnant women of South Asian ethnicity had a substantially lower B-12 status than pregnant women of European ethnicity. The 30%–36% prevalence of South Asian women classified as B-12 deficient (defined as total B-12 <148 pmol/L) was also higher compared to recent reports of Canadian 178  pregnancy cohorts with mixed ethnicities with a 0% prevalence of B-12 deficiency (defined as holoTC <35 pmol/L) in the 1st and 2nd trimester (221) and  10%–35% prevalence of B-12 deficiency in the 2nd trimester and at delivery (defined as total B-12 <148 pmol/L) (156,181). A high prevalence of B-12 deficiency in South Asian populations has previously been reported in Canada and the US ranging from 7% (total B-12 <118 pmol/L) to up to 60% (total B-12 <180 pmol/L) in adult men and women (169,190–192). Intake of animal-source foods and supplemental B-12 intake have been identified as predictors of B-12 status in young adult women of South Asian and European ethnicity (169). A study in New Zealand found that the 48% prevalence of B-12 inadequacy (total B-12 <222 pmol/L) in 62 women of South Asian ethnicity was related to inadequate dietary B-12 intake (<2.4 µg/d, assessed by food frequency questionnaire), especially by following diets avoiding some animal-source foods, such as lacto- or lactoovo-vegetarian diets (274). The B-12 status in these South Asian women was improved after 6-mo supplementation with 6 µg/d of B-12, however, not after dietary advice. These findings suggest that the high prevalence of B-12 deficiency in women of South Asian ethnicity may be related to their low dietary intakes of B-12. In Canada, however, Quay et al. (169) found no significant difference in supplement use or dietary B-12 intake between reproductive-aged women of South Asian and European ethnicity, while there was a tendency for a lower B-12 status in women of South Asian ethnicity. In conclusion, pregnant women of South Asian ethnicity living in Vancouver, Canada, have a lower B-12 status as assessed by multiple biomarkers; however, my data do not allow concluding whether low dietary B-12 intake explains this observation. Also, current literature is inconsistent regarding whether dietary B-12 intake is the strongest predictor for the observed difference in B-12 status and prevalence of B-12 deficiency between ethnic groups. 179  Disturbances in B-12 biomarker concentrations have been associated with certain genotypes (124,168,276). As such, genetic differences between ethnicities may contribute to some of the differences in B-12 status between South Asians and Europeans. However, with the exception of genes related to inborn errors of B-12 metabolism, only minor differences in B-12 biomarker concentrations have been linked to genetic polymorphisms (134). The MTHFR C677T variant is the most commonly studied genotype in relation to B-vitamin metabolism. The homozygous TT genotype has been related to a greater risk of B-12 deficiency (total B-12 <148 pmol/L) in healthy adults [odds ratio (OR): 1.78; 95% CI: 1.25; 2.54] (168). Yet, the prevalence of the TT and CT allele is significantly lower in South Asians than Europeans (169,308) and has not been associated with an increased prevalence of lower total B-12 concentrations (169), elevated tHcy concentrations or NTD-affected pregnancies in South Asians (308). Additionally, a higher prevalence of the G allele of the FUT2 A893G genotype has been reported in South Asians compared to Europeans (169). The G allele of the FUT2 A893G genotype has been associated with lower total B-12 concentrations in South Asians in 1 study of 1146 individuals in India (276). This finding, however, was not observed in 55 young adult women of South Asian ethnicity residing in Vancouver, Canada (169). Thus, further research is needed to determine if the FUT2 A893G genotype is a significant predictor of the low total B-12 concentrations observed in South Asian populations, including the pregnant women in the present study. Further, variants of the transcobalamin gene (TCN2 C677G and A67G) have been associated with low circulating holoTC concentrations (170,171) and altered tHcy (172) and MMA (173) concentrations, although not consistently (174). Recently, genome-wide sequencing of 2,210 healthy Irish adults revealed that 12% of the variability in circulating MMA concentrations is accounted for by mutations in HIBCH, the 180  gene encoding for the enzyme 3-hydroxyisobutyryl-CoA hydrolase, which is part of the valine metabolism, and in the gene encoding for the acetyl-CoA synthase (ACSF3) (124). However, the prevalence of these genetic variants in South Asians has not yet been studied. Thus, it is possible that the substantial differences in B-12 status between South Asian and European pregnant women can be explained by certain genetic variants. However, further research is needed exploring the prevalence and distribution of genetic variants associated with disturbances in B-12 status in South Asian compared to other populations. My findings suggest that women of South Asian ethnicity, Canada’s largest ethnic minority (48), may be at increased risk for adverse health consequences related to B-12 deficiency during pregnancy but the predictors are not yet known. South Asian newborns are more likely small-for-gestational age, preterm, or have a low birth weight (30,309). The rate of NTD-affected pregnancies is 10-times higher in India compared to Canada where the incidence is 4.1 NTDs per 10,000 births (44) (Chapter 1.2.3), despite folic acid fortification programs in both countries. A higher prevalence of low birth weight and preterm birth has also been reported in South Asian populations compared to other ethnicities in the US (257). In the present study, South Asian women had a significantly higher prevalence of low birth weight-infants (3.3%) and a tendency for a higher prevalence of preterm infants (4.2%) compared to European women (0.62% and 2.5%, respectively). Yet, given the limitations in data collection in the present study, i.e. missing information on maternal weight gain during pregnancy and other confounding factors, a correlation with maternal B-12 status cannot be tested. Maternal B-12 deficiency during pregnancy, however, is associated with such adverse pregnancy outcomes, including small-for-gestational age (55), low birth weight (215,249), preterm birth (310), and NTDs (38,39). Taken together, these observations lead to the hypothesis that the 181  higher prevalence of adverse health consequences in South Asian pregnant women and their newborns in the present and other studies may be, in part, due to the higher prevalence of B-12 deficiency. Targeted interventions to improve B-12 status in pregnant women of South Asian ethnicity are warranted to investigate its impact on adverse pregnancy outcomes and improve maternal and infant health in Canada. My findings of a high prevalence of B-12 deficiency in South Asian pregnant women is especially of concern in countries where folic acid intake and folate status are high, and predominantly so in pregnant women, as is the case in Canada (195,221). In South Asian women living in India, a B-12/folate imbalance during pregnancy has been associated with small-for-gestational age offspring (55) and diabetes in the offspring (59). In a mouse model, it has been suggested to amplify metabolic consequences of B-12 deficiency (311). Folate status of all women in the present study of pregnant Vancouver women was high (>85% had serum folate concentrations >45.3 nmol/L). These results, however, should be interpreted with caution because serum folate concentrations are known to fluctuate widely with food intake, and in this study the measurements were conducted in non-fasting blood samples. The preferred biomarker for long-term folate status is RBC folate; however, this type of specimen was unavailable for the present research. Yet, the present study showed that South Asian pregnant women in Vancouver have a high prevalence of B-12 deficiency and high circulating folate concentration, so might be at increased risk for adverse pregnancy outcomes associated with an imbalanced perinatal B-12 and folate status. Furthermore, I determined that infants (aged 6–12 mo) living in rural Indonesia have a 27% prevalence of B-12 deficiency, using serum total B-12 as a biomarker with an age-specific reference value. This is the first study to report on B-12 deficiency in Indonesia and is, to my 182  knowledge, 1 of 3 recent reports from Southeast Asia (312,313). The prevalence of B-12 deficiency (total B-12 <150 pmol/L) was substantially lower in adolescent girls (<1%), and in reproductive-aged women (4%) and pregnant women (3%) living in Myanmar and rural Cambodia, respectively (312,313). The 38% prevalence of inadequate B-12 status (total B-12 <220 pmol/L) in the Indonesian infants in the present study was comparable to the prevalence of inadequate B-12 status observed in infants in other LMIC (39-43%), including Nepal, Bangladesh, and Guatemala (213,219,295). These findings suggest that B-12 deficiency is a potential health concern in Indonesia. My research contributes to the knowledge that B-12 deficiency is prevalent in breastfed infants living in LMIC (293), and is likely predicted by maternal B-12 deficiency (219). Improving maternal B-12 status during lactation appears to be important for decreasing infantile B-12 deficiency. However, earlier interventions during fetal and infant development, i.e. during pregnancy and at birth, have also been shown to improve infant B-12 status (35). Infants of B-12 deficient mothers are born with low B-12 stores, which will be depleted during the 1st year of life if untreated (32). Breastmilk of B-12 deficient women provides inadequate amounts of B-12 to the infant (212). Thus, interventions to improve B-12 status in infants in LMIC should not only focus on infants but also include pregnant and lactating women (35,213). Additionally, significant correlations between B-12 intake from complementary foods and infant B-12 status have been shown (219). Thus, inclusion of B-12 rich foods during complementary feeding may also be a strategy for improving infant B-12 status in LMIC. Future research is needed to identify determinants of B-12 status in the present study in infants living in rural Indonesia, focusing on a comprehensive assessment of maternal B-12 deficiency, breastfeeding and complementary feeding practices. 183  This work contributed new knowledge about the prevalence of B-12 deficiency in vulnerable population groups at stages of rapid growth including infants and pregnant women. Pregnant women of South Asian ethnicity living in Vancouver, Canada, and rural Indonesian infants were identified as at-risk populations. 6.3. Limitations of the Studies While specific limitations are discussed within the chapters, the following section summarizes the 2 major limitations of my research: missing clinical follow-up data and limited data on predictors of B-12 status. Individuals in the present studies were identified as B-12 deficient based on biomarker concentrations only. Reference intervals were used to categorize participants’ B-12 status, given the lack of population-specific validated cutoffs. Thus, study participants might have been misclassified. Also, participants’ B-12 deficiency was not confirmed by clinical outcomes, i.e. hematological or neurological measures. The clinical hallmark for B-12 deficiency is anemia (2,82). However, adverse health outcomes related to B-12 status have been reported in pregnant women and infants without anemia (7,32). No clinical symptom has yet been identified as a single trait of inadequate B-12 status in pregnant women, newborns, or infants. Impaired neurocognitive development and function is the adverse health outcome which has been speculated to relate to inadequate perinatal or infantile B-12 status (7,32). However, results of randomized-controlled trials investigating the relationship between supplemental B-12 during pregnancy and cognition and developmental hallmarks in the offspring are inconclusive (74,80). Additional studies are needed to determine novel 184  functional, physiological and/or clinical outcomes for perinatal, neonatal, and infantile B-12 status to allow confirming B-12 deficiency in these populations.  Furthermore, no detailed conclusions regarding predictors of B-12 deficiency can be drawn from the studies. B-12 status can be influenced by dietary B-12 intake, B-12 absorption, genetic variants, or a combination of all, among other confounding factors (6,8). Assessment of dietary B-12 intakes can be challenging, especially in ethnic minorities and LMIC. Validated dietary assessment tools (314), and complete nutrient database files are not available for all populations and countries. Neither the study located in Indonesia (Chapter 5) nor the cohort study in Vancouver (Chapter 4) did assess any dietary, genetic or other validated health variables, such as gastro-intestinal disease. Further research is needed to identify predictors of the observed high prevalence of B-12 deficiency in infants living in rural Indonesia and pregnant women of South Asian ethnicity residing in Metro Vancouver. Such research is necessary to allow for future targeted intervention, e.g. supplementation, fortification or dietary advice, and inform policies. 6.4. Future Directions 6.4.1. Evaluation of Reference Values and Alternate Indicators for Vitamin B-12 Status in Pregnant Women and Newborns Pregnancy-specific cutoffs Several studies have suggested reference values, computed from the 95% CI of a healthy population, to determine B-12 status in pregnant women (Table 1-5). Yet, no studies have been undertaken to evaluate and validate these suggested reference values against clinical 185  symptoms in pregnant women, newborns, or infants. Such evaluation is necessary to establish reliable and meaningful cutoffs linked to health outcomes, and is discussed below (12). Perinatal B-12 deficiency has been associated with impaired fetal development, such as low birth weight, intra-uterine growth restriction, small-for-gestational age, NTDs, and other congenital anomalies as well as low fetal B-12 stores (Chapter 1.2.1). The diagnostic capacities of B-12 biomarkers to identify women with impaired fetal development need to be determined to identify valid and clinically meaningful biomarkers and related cutoffs in pregnant women. Such studies would include evaluating the utility of each biomarker as well as the reference values suggested in this and previous work against defined clinical and functional outcomes using case-control studies or ROC analyses. To date, 2 case-control studies involving NTD-affected pregnancies have been undertaken and suggested cutoffs indicative of B-12 deficiency in women commencing pregnancy and during early pregnancy (<28 d) (38,39). However, the work is limited to 2 studies assessing 1 biomarker each, i.e. holoTC and total B-12, respectively. The work has yet to be repeated in other studies including multiple biomarkers of B-12 status and other developmental outcomes that have been inversely associated with perinatal B-12 status. Randomized controlled trials Additionally, no randomized controlled trials, the gold standard clinical study design to determine causality, have yet been undertaken to confirm associations between maternal B-12 inadequacy during pregnancy and adverse pregnancy outcomes, e.g. NTDs. Given the recent public interest, broader research into the cause and prevention of birth defects has been suggested to improve infant and general public health (315). This public trend may be used to 186  further explore the associations between perinatal B-12 intake and status with NTDs and other congenital anomalies. Using a trial, randomizing women to either a B-12 supplement or placebo with dietary advice, given ethical considerations, in a population with a high prevalence of NTDs or other congenital anomalies, such as Northern China (316,317), would be the next step to elucidate the causality between perinatal B-12 intake or status and birth defects. Ideally women would be enrolled before pregnancy to reach serum total B-12 and holoTC concentrations associated with a decreased risk of a NTD-affected pregnancy [>220 pmol/L (39) and >55 pmol/L (38), respectively] at the time of the neural tube closure (28 d of gestation). However, given the low rate of NTDs even in regions with a high prevalence of birth defects, e.g. 44 cases per 13,956 still- and live births in Shanxi Province, Northern China, in 2014 (317), it has to be acknowledged that such trials require much human and financial resources. Yet, they would be crucial to provide information on the adverse health outcomes caused by perinatal B-12 deficiency and identify biomarker cutoffs associated with such clinically meaningful adverse health outcomes. Alternate indicators of vitamin B-12 status in pregnant women and newborns Lastly, given the limited diagnostic utility of the current biomarkers of B-12 status to identify pregnant women at risk for having a newborn with B-12 deficiency, alternate indicators of B-12 status should be explored. Several algorithms as well as alternate biomarkers have been suggested to have advantages in measuring B-12 status (8,129,318). Fedosov et al. (127) proposed a mathematical model combining, ideally, all 4 common B-12 biomarkers, i.e. total B-12, holoTC, MMA, and tHcy. The model was shown to be a sensitive indicator of B-12 status in adults with cognitive impairment (128) and anemia (129) due to B-12 deficiency. It has been proposed to be a more sensitive and specific indicator of B-12 status than any of 187  the 4 biomarkers alone. Further, formate as a biomarker of B-12 status (318) during pregnancy may be explored. Formate is involved in the one-carbon metabolism as a methyl-donor required for the B-12-dependent remethylation of homocysteine to methionine (282). Plasma and urinary formate concentrations were found to be elevated in the case of B-12 deficiency in rats (319). Either indicator of B-12 status, i.e. the mathematical model and formate, should be evaluated against clinical and functional outcomes in mother and offspring. Such studies may provide insight into the more valid biomarker for B-12 status during pregnancy. In addition to DBS MMA, acylcarnitines, namely propionylcarnitine (C3), its ratio with acetylcarnitine (C3/C2) or palmitoylcarnitine (C3/C16), and heptadecanoylcarnitine (C17), have been suggested as indicators of B-12 status in newborns (65,281,320). The disturbance of the odd-chain fatty acid metabolism through B-12 deficiency has been hypothesized to lead to an accumulation of propionyl-CoA, an educt of the MMA synthesis, which favors the synthesis of 2-methyl branched chain fatty acids (acylcarnitines) (321). Acylcarnitines are routinely quantified during the newborn screening in Canada (132). Exploring the correlation between maternal B-12 status during pregnancy and neonatal acylcarnitines concentration or ratio may reveal further possibilities to test for B-12 inadequacy in newborns in routinely collected DBSs. Age-specific cutoffs for infants Reference values of B-12 biomarkers in infants also need to be evaluated, and the impact of breastfeeding on infantile B-12 status needs to be better understood. Breastfed infants have a significantly lower B-12 status compared to formula-fed infants (163). It has been suggested that the inadequate B-12 status seen in breastfed infants is associated with poor 188  infant cognitive development (73). Yet, breastfeeding overall appears to be associated with improved infant cognitive development (322,323). Current reference intervals were obtained from mostly breastfed infants (162). Future research is needed to determine the sensitivity and specificity of current reference values against validated measures of cognitive development in infants. 6.4.2. Predictors of Vitamin B-12 Deficiency in Pregnant Women of South Asian Ethnicity in Canada In light of the 30%–36% prevalence of South Asian pregnant women being classified as B-12 deficient, more research is warranted to identifying predictors of B-12 deficiency in the largest ethnic minority in Canada. Possible predictors include low dietary intake of animal-source foods (188), low supplement use (169), and genetic variants (276), however, findings are inconclusive. Given that data collection on dietary intake or supplement use was not feasible in the present study of pregnant women of South Asian ethnicity residing in Vancouver, no conclusions can yet be drawn about whether the higher prevalence of perinatal B-12 deficiency in women of South Asian ethnicity compared to those of European ethnicity is related to any of these predictors. Future research is needed to determine the predictors of perinatal B-12 deficiency in South Asian women living in Vancouver to allow for targeted interventions and reduce and prevent the high prevalence of B-12 deficiency and potentially related adverse health outcomes in South Asians living in Canada and world-wide.  6.4.3. Targeted Interventions In my work, I have introduced DBSs as a screening tool for B-12 status in newborns and remote populations. DBS MMA allows for convenient analysis of B-12 status. Thus, DBS 189  MMA may be used for targeted screening in at-risk populations. Inclusion of DBS MMA quantification in the newborn screening program in Canada would allow for testing of B-12 deficiency in this vulnerable population group. Screening for neonatal B-12 deficiency has been discussed as a means to prevent severe neurological damage (64). Currently, DBS MMA is used as a 2nd-tier test in the screening for inborn errors of B-12 metabolism. However, this practice is not sensitive enough to diagnose newborns with secondary B-12 deficiency. Cost-benefit analyses are necessary to identify criteria by which DBS MMA will be quantified in a routine 2nd-tier test for neonatal B-12 deficiency second to maternal deficiency. Given the 27% prevalence of B-12 deficiency, future studies are needed to improve B-12 status of breastfed infants in Indonesia. B-12 supplementation (50 µg/d) of pregnant as well as lactating women was shown to significantly increase B-12 status in mothers and their infants in India (35,213). However, more culturally appropriate methods than supplementation may be necessary to efficiently improve micronutrient status in Indonesia and other countries. Multiple studies have demonstrated the efficacy and efficiency of micronutrient powders and fortification programs to reduce infant nutrient deficiencies in LMIC (324,325). 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