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Vitamin B6 status of young and older adult women in Metro Vancouver Ho, Chia-ling 2017

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VITAMIN B6 STATUS OF YOUNG AND OLDER ADULT WOMEN IN METRO VANCOUVER by  Chia-ling Ho  B.Sc., Taipei Medical University, 2014  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Human Nutrition)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2017  © Chia-ling Ho, 2017 ii Abstract Vitamin B6 (B6) plays an essential role in the metabolism of amino acids, synthesis of neurotransmitters, and regulation of energy homeostasis. B6 deficiency, plasma pyridoxal 5’-phosphate (PLP) concentration <20 nmol/L, has been associated with impaired immune responses and depression. Suboptimal B6 status, plasma PLP concentration between 20−30 nmol/L, has been associated with cardiovascular disease and cancer. A 19.4% prevalence of inadequate dietary B6 intake was reported among women aged 51−70 years and 9.6% women aged 19−30 years in the Canadian Community Health Survey 2004. However, there are very few data on biochemical B6 status among Canadian women. This thesis aimed to assess B6 status and to identify predictors of B6 status, in young and older adult women in Metro Vancouver, Canada.  Vitamin B6 status in young adult women was assessed from an existing, descriptive, cross-sectional study with a convenience sample of 202 women aged 19−35 years in Metro Vancouver. Another descriptive, cross-sectional study was conducted in 223 older adult women aged 51−70 years in Metro Vancouver recruited by convenience sampling. B6 status was determined by fasting plasma PLP concentrations. Information on demographic and lifestyle characteristics and dietary intake was collected through questionnaires.  The prevalence of B6 deficiency was 1.5% in young and 1.4% in older adult women. Suboptimal B6 status was 10.9% in young and 8.1% in older adult women. In both samples, the participants had high education levels and about 30% used B6-containing supplements. Body mass index, dietary B6 intake, and the use of supplemental B6 were significant predictors of plasma PLP concentrations in both samples. South Asian ethnicity was identified as a negative iii predictor of plasma PLP concentration in young adult women but a positive predictor in older adult women. Suboptimal B6 status was prevalent in convenience samples of young and older adult women in Metro Vancouver. Ethnicity, dietary B6 intake and the use of supplemental B6 should be considered as covariates for predicting B6 status in future studies. Considering the high socioeconomic status of the participants in this thesis, there is a need to investigate B6 status in a representative sample of Canadian women. iv Lay Summary Vitamin B6 (B6) is important for brain health, cell growth, and energy levels. Low B6 status has been linked to early pregnancy loss, cardiovascular diseases and cancers. However, information on blood levels of B6 in Canadian women is limited. This thesis aimed to assess the blood levels of B6 and the prevalence of low B6 status in young and older adult women in Metro Vancouver. The prevalence of low B6 status was 12% and 10% in women aged 19−35 and 51−70 years, respectively. Women who had lower body mass index, reported higher dietary B6 intake, and used supplements containing B6, were found to have higher blood B6 levels. We are the first to report the prevalence of low B6 status in non-pregnant, young adult women and in older adult women in Canada. This thesis warrants more research on possible prevention strategies of low B6 status among Canadian women. v Preface This thesis is my original work carried out under the supervision of Dr. Yvonne Lamers, with guidance and support from my committee members Dr. Angela Devlin and Dr. Jennifer Black. My thesis consists of two studies.  A version of Chapter 3 is published (Ho C-L, Quay TA, Devlin AM, and Lamers Y. Prevalence and Predictors of Low Vitamin B6 Status in Healthy Young Adult Women in Metro Vancouver. Nutrients. 2016 Sep; 8(9): 538) [1]. The study in Chapter 3 is a secondary analysis using the data from a cross-sectional study designed and conducted by Dr. Yvonne Lamers and Teo Quay. I performed the blood analysis for measurement of vitamin B6 status in Dr. Lamers’ laboratory with the help of a research assistant, Benny Chan. I was also responsible for the data analysis and interpretation as well as the preparation of the first manuscript and chapter draft. Dr. Angela Devlin contributed to manuscript edits. Dr. Yvonne Lamers acted as the primary investigator and supervisor in this project, and contributed to data interpretation and manuscript and chapter revisions. I presented preliminary results of this study at the University of British Columbia (UBC) Faculty of Land and Food Systems Graduate Student Conference in March 2015. I disseminated the research outcomes of this secondary analysis in form of a poster presentation at the Experimental Biology Meeting / American Society for Nutrition Annual Meeting in April 2016 in San Diego, California (FASEB J 2016; 30 (Suppl 1): 1171.6). My abstract was selected as a finalist in the Emerging Leaders in Nutrition Science Poster Competition. The original study including this secondary analysis received ethical approval from the UBC Clinical Research Ethics Board (#H11-01216), the Vancouver Coastal Health Research Ethics Board (#V11-01216), and the Fraser Health Research Ethics Board (#FHREB 2013-016). vi This research was funded by the “Food, Nutrition and Health Vitamin Research Fund”, Faculty of Land and Food Systems, UBC. Chapter 4 is an original study designed by Dr. Yvonne Lamers and myself. The research team was comprised of myself, research assistants, and undergraduate research volunteers. I played an integral role in leading the cross-sectional study described in Chapter 4. I was involved in all aspects of the research project from study design to participant recruitment, conduct of study visits, and data analysis. I recruited and enrolled participants, and completed assessments and data collection including questionnaire administration and anthropometric measurements. I performed blood processing with the help of research assistants in Dr. Lamers’ laboratory (FNH1, UBC) and in the Analytical Core for Metabolomics and Nutrition (BCCHRI2). The Alberta Health Services3 carried out the analysis of the Canadian Diet History Questionnaire. I conducted the majority of the laboratory experiments involving biomarker analysis with the help of research assistants, and I completed the data cleaning of all questionnaire data with the help of undergraduate research volunteers. I performed all statistical analyses. Dr. Yvonne Lamers acted as primary investigator and supervisor of this research with the oversight on study conduct and biomarker analyses, and contributed to data interpretation and chapter revisions. I presented this research at the UBC Faculty of Land and Food Systems Graduate Student Conference in March 2017, where I scored second in the poster competition and also won the People’s Choice Award for Best Poster. I also disseminated this research in form of a poster presentation at the Annual Meeting of the Canadian Nutrition Society in May 2017 in Montreal, Quebec (APNM                                                    1"Human"Nutrition"and"Vitamin"Metabolism"Research"Group"(www.vitamins.landfood.ubc.ca);"Food,"Nutrition,"and"Health"Program;"Faculty"of"Land"and"Food"Systems;"The"University"of"British"Columbia;"Vancouver"BC,"Canada"2"British"Columbia"Children’s"Hospital"Research"Institute;"Vancouver"BC,"Canada"3"CancerControl"Alberta,"Department"of"Cancer"Epidemiology"and"Prevention"Research;"Calgary"AB,"Canada"vii 2017;42:S18).  The UBC Clinical Research Ethics Board (#H15-01937) and the Vancouver Coastal Health Ethics Board (#V15-01937) have approved this study. viii Table of Contents Abstract .......................................................................................................................................... ii"Lay Summary ............................................................................................................................... iv"Preface .............................................................................................................................................v"Table of Contents ....................................................................................................................... viii"List of Tables ............................................................................................................................... xiv"List of Figures ............................................................................................................................. xvi"List of Abbreviations ................................................................................................................. xvii"Acknowledgements .................................................................................................................. xviii"Chapter 1: Overview ....................................................................................................................... 1"Chapter 2: Literature Review .......................................................................................................... 4"2.1" Preamble ..................................................................................................................... 4"2.2" Metabolic Forms of Vitamin B6 ................................................................................. 5"2.3" Functions of Vitamin B6 ............................................................................................. 7"2.4" Indicators for Vitamin B6 Status Assessment ........................................................... 10"2.4.1" Direct Indicators of Vitamin B6 Status ............................................................. 10"2.4.2" Functional Indicators of Vitamin B6 Status ...................................................... 13"2.5" Sources and Recommendation of Dietary Vitamin B6 ............................................. 17"2.5.1" Food Sources of Vitamin B6 ............................................................................. 17"2.5.2" Recommendations for Dietary Vitamin B6 Intake ............................................ 17"2.6" Determinants and Predictors of Biochemical Vitamin B6 Status ............................. 19"2.6.1" Dietary Vitamin B6 ........................................................................................... 21"2.6.2" Supplement Use ................................................................................................ 22"ix 2.6.3" Oral Contraceptive Use and Hormone Therapy ................................................ 23"2.6.4" Physical Activity ............................................................................................... 26"2.6.5" Smoking ............................................................................................................ 27"2.6.6" Socioeconomic Status ....................................................................................... 29"2.6.7" Ethnicity ............................................................................................................ 30"2.6.8" Alcohol Consumption ....................................................................................... 32"2.6.9" Body Mass Index .............................................................................................. 33"2.7" Cut-offs for Vitamin B6 Status and Health Consequences Associated with Vitamin B6 Deficiency and Suboptimal B6 Status ............................................................................. 35"2.7.1" Categories of Vitamin B6 Status ....................................................................... 35"2.7.2" Clinical Consequences of Vitamin B6 Deficiency ............................................ 36"2.8" Health Implications Associated with Low Vitamin B6 Status .................................. 36"2.8.1" Cardiovascular Diseases ................................................................................... 36"2.8.2" Breast Cancer .................................................................................................... 37"2.8.3" Colorectal Cancer .............................................................................................. 39"2.8.4" Lung Cancer ...................................................................................................... 40"2.8.5" Inflammation ..................................................................................................... 41"2.9" Low Vitamin B6 Status in Women ............................................................................ 42"2.9.1" Women of Childbearing Age ............................................................................ 42"2.9.2" Older Adult Women .......................................................................................... 44"2.10" Vitamin B6 Status of Canadians ............................................................................... 45"2.11" Summary and Rationale ............................................................................................ 47"x Chapter 3: Prevalence and Predictors of Low Vitamin B6 Status in Healthy Young Adult Women in Metro Vancouver ....................................................................................................................... 49"3.1" Introduction ............................................................................................................... 49"3.2" Methods..................................................................................................................... 50"3.2.1" Study Design and Participants .......................................................................... 50"3.2.2" Biospecimen and Data Collection ..................................................................... 51"3.2.3" Laboratory Analysis .......................................................................................... 52"3.2.4" Final Variables Included in the Analysis ........................................................... 53"3.2.5" Statistical Analyses ........................................................................................... 56"3.2.5.1" Preliminary Univariate Analysis ................................................................... 56"3.2.5.2" Primary Objective ......................................................................................... 56"3.2.5.3" Secondary Objective ..................................................................................... 57"3.3" Results ....................................................................................................................... 58"3.3.1" Participant Characteristics ................................................................................ 58"3.3.2" Dietary Intakes and Associations with Food Sources ....................................... 60"3.3.3" Primary Objective: Prevalence of Vitamin B6 Deficiency and Suboptimal Vitamin B6 Status ............................................................................................................. 62"3.3.4" Secondary Objective: Predictors of Vitamin B6 Status .................................... 63"3.3.4.1" Bivariate Analysis ......................................................................................... 63"3.3.4.2" Multivariable Regression Model ................................................................... 67"3.4" Discussion ................................................................................................................. 68"3.4.1" Prevalence of Vitamin B6 Deficiency and Suboptimal Vitamin B6 Status ...... 68"3.4.2" Predictors of Plasma Pyridoxal 5’-phosphate ................................................... 71"xi 3.4.2.1" Use of Supplemental Vitamin B6 ................................................................. 71"3.4.2.2" Dietary Vitamin B6 Intake ............................................................................ 72"3.4.2.3" Ethnicity ........................................................................................................ 72"3.4.2.4" Body Mass Index .......................................................................................... 73"3.4.2.5" Other Variables .............................................................................................. 74"3.4.3" Strengths and Limitations ................................................................................. 75"3.5" Conclusion ................................................................................................................ 77"Chapter 4: Vitamin B6 Status in Older Adult Women in Metro Vancouver ................................. 78"4.1" Introduction ............................................................................................................... 78"4.2" Methods..................................................................................................................... 79"4.2.1" Study Design ..................................................................................................... 79"4.2.2" Study Participants ............................................................................................. 80"4.2.2.1" Sample Size ................................................................................................... 80"4.2.2.2" Inclusion and Exclusion criteria .................................................................... 80"4.2.3" Recruitment ....................................................................................................... 81"4.2.4" Study Visit Procedure ....................................................................................... 82"4.2.5" Demographic and Food Frequency Questionnaires .......................................... 85"4.2.6" Laboratory Methods .......................................................................................... 87"4.2.6.1" Initial Blood Sample Processing ................................................................... 87"4.2.6.2" Biochemical Vitamin B6 Status Assessment ................................................ 91"4.2.7" Final Variables Included in Analysis ................................................................. 92"4.2.8" Statistical Analyses ........................................................................................... 94"4.3" Result ........................................................................................................................ 96"xii 4.3.1" Participants Characteristics ............................................................................... 96"4.3.2" Dietary Intakes .................................................................................................. 99"4.3.3" Primary Objective: prevalence of B6 deficiency and suboptimal status .......... 99"4.3.4" Secondary Objective: Predictors of Vitamin B6 Status .................................. 100"4.3.4.1" Bivariate Analysis ....................................................................................... 100"4.3.4.2" Multivariable Regression Model ................................................................. 104"4.4" Discussion ............................................................................................................... 105"4.4.1" Prevalence of Vitamin B6 Deficiency and Suboptimal Vitamin B6 Status .... 105"4.4.2" Predictors of Plasma Pyridoxal 5’-phosphate Concentration ......................... 107"4.4.2.1" Supplemental Vitamin B6 Intake ................................................................ 107"4.4.2.2" Dietary Vitamin B6 Intake .......................................................................... 108"4.4.2.3" Ethnicity ...................................................................................................... 108"4.4.2.4" Body Mass Index ........................................................................................ 109"4.4.2.5" Other Variables ............................................................................................ 110"4.4.3" Strengths and Limitations ................................................................................ 111"4.5" Conclusion .............................................................................................................. 113"Chapter 5: General Discussion and Conclusion .......................................................................... 114"5.1" Summary ................................................................................................................. 114"5.2" General Discussion ................................................................................................. 116"5.2.1" Vitamin B6 Status in Young and Older Adult Women in Metro Vancouver ... 116"5.2.2" Limitations ...................................................................................................... 118"5.2.3" Future Directions ............................................................................................ 119"5.3" Conclusion .............................................................................................................. 120"xiii Bibliography ...............................................................................................................................122"Appendices ..................................................................................................................................145""xiv List of Tables Table 2-1 Summary of Vitamin B6 Status Indicators ................................................................... 16"Table 2-2 Dietary Reference Intakes for Vitamin B6 ................................................................... 19"Table 2-3 Large-scale Studies Investigating Factors Affecting Vitamin B6 Status ...................... 20"Table 2-4 Usual Dietary Vitamin B6 Intake from Food Sources in Canadian Adult Women, Canadian Community Household Survey 2004 ............................................................................ 47"Table 3-1 Variables and Coding Method Included in the Analysis of Vitamin B6 Status in Young Adult Women in Metro Vancouver ............................................................................................... 55"Table 3-2 Demographic, Anthropometric, and Lifestyle Characteristics in Young Adult Women in Metro Vancouver ........................................................................................................................... 59"Table 3-3 Lifestyle Characteristics in Young Adult Women in Metro Vancouver ........................ 60"Table 3-4Dietary Intake in Young Adult Women in Metro Vancouver ........................................ 61"Table 3-5Food Sources of Vitamin B6 in Young Adult Women in Metro Vancouver ................. 61"Table 3-6Plasma Pyridoxal 5’-phosphate Concentration and Prevalence of Vitamin B6 Deficiency, Suboptimal Status, and Adequacy in Young Adult Women in Metro Vancouver ...... 63"Table 3-7Plasma Pyridoxal 5’- phosphate Concentration by Categories of Demographic and Lifestyle Factors in Young Adult Women in Metro Vancouver .................................................... 65"Table 3-8 Plasma Pyridoxal 5’-phosphate Concentration by Categories of Demographic and Lifestyle Factors in Non-users of Supplemental Vitamin B6 in Young Adult Women in Metro Vancouver ..................................................................................................................................... 66"Table 3-9 Unadjusted Association of Continuous Variables with Plasma Pyridoxal 5’-phosphate in Young Adult Women in Metro Vancouver ................................................................................ 67"xv Table 3-10 Predictors of Plasma Pyridoxal 5’-phosphate Concentration in Young Adult Women in Metro Vancouver ....................................................................................................................... 68"Table 4-1 Calculations for Selected Variables in Vitamin B6 Status of Older Adult Women in Metro Vancouver ........................................................................................................................... 86"Table 4-2 Variables and Coding Method Included in Vitamin B6 Status of Older Adult Women in Metro Vancouver ........................................................................................................................... 93"Table 4-3 General Participant Characteristics of Older Adult Women in Metro Vancouver ........ 97"Table 4-4 Lifestyle Characteristics of Older Adult Women in Metro Vancouver ......................... 98"Table 4-5 Dietary Intake of Older Adult Women in Metro Vancouver ......................................... 99"Table 4-6 Plasma Pyridoxal 5’-phosphate Concentration and Prevalence of Vitamin B6 Deficiency, Suboptimal Status, and Adequacy in Older Adult Women in Metro Vancouver ....... 99"Table 4-7Plasma Pyridoxal 5’-phosphate Concentration by Categories of Demographic and Lifestyle Factors in Older Adult Women in Metro Vancouver ................................................... 102"Table 4-8Plasma Pyridoxal 5’-phosphate Concentration by Categories of Demographic and Lifestyle Factors in Non-users of Supplemental Vitamin B6 in Older Adult Women in Metro Vancouver ................................................................................................................................... 103"Table 4-9 Unadjusted Association of Continuous Variables with Plasma Pyridoxal 5’-phosphate Concentration in Older Adult Women in Metro Vancouver ........................................................ 104"Table 4-10 Predictors of Plasma Pyridoxal 5’-phosphate Concentration in Older Adult Women in Metro Vancouver ......................................................................................................................... 105"Table 5-1 Summary of Vitamin B6 Status in Young and Older Adult Women in Metro Vancouver..................................................................................................................................................... 115" xvi List of Figures Figure 2-1 Conversion and Catabolism of Vitamin B6 Vitamers ................................................... 7"Figure 2-2 Roles of Vitamin B6 in One Carbon Metabolism ......................................................... 9"Figure 2-3 Tryptophan Catabolism through Kynurenine Pathway ............................................... 10"Figure 3-1 Distribution of Plasma Pyridoxal 5’-phosphate Concentration in Young Adult Women in Metro Vancouver ....................................................................................................................... 62"Figure 4-1 Procedure for the Study Visits ..................................................................................... 84"Figure 4-2 Procedure for Initial Blood Sample Processing .......................................................... 90"      xvii List of Abbreviations B6= vitamin B6 BC= British Columbia BMI= body mass index CCHS= Canadian Community Health Survey C-DHQ= Canadian Diet History Questionnaire CI= confidence interval cm= centimeter DHQ= Diet History Questionnaire EAR= estimated average requirement FFQ= food frequency questionnaire g= gram Hb= hemoglobin HPLC= high performance liquid chromatography IPAQ= International Physical Activity Questionnaire kcal= kilocalories kg= kilogram LC/MS-MS= liquid chromatography-tandem mass spectrometry µg= microgram mg= milligram NHANES= National Health and Nutrition Examination Survey OC= oral contraceptive OR= odds ratio PBS= phosphate buffered saline PLP= pyridoxal-5’ phosphate RDA= recommended daily allowance RR= relative risk SD= standard deviation UBC= The University of British Columbia US= United States xviii Acknowledgements First and foremost, I would like to thank all women who voluntarily participated in this research study. Without their enthusiasm to participate in research, this thesis would not have been completed.  Next, I like to extend my thanks to my supervisor, Dr. Yvonne Lamers. Throughout this process, I’ve improved my communication skills in both writing and orally, and critical thinking. You always provide valuable insight and unconditional support. I would also like to thank my committee members Dr. Angela Devlin, and Dr. Jennifer Black for supporting me over the past few years. I’m grateful for your support and valuable feedback. To my supportive lab mates and colleagues Theresa, Fernanda, Matthew, Amy, Dalal, Vanessa, Zubair, Aviva, Claire, Abeer, and Ellie for making this positive learning experience memorable.  Special thanks to Matthew for his great support during the last period of my study helping with the recruitment, study visits, lab work and data entry. I would like to thank my undergraduate research volunteers Christine, Grace, Sylvia, Mirah, and Alex for their help on study days and data entry. Also, thanks to the Innis Lab, especially Roger Dyer and Janette King, for contributing laboratory space. Thanks to the Alberta Health Services for their assistance in dietary data analysis. To my friends Alice, Shirley, and Helen – thank you for pulling me out of school and showing me this city with good food. To Victor, thank you for your unconditional support, for always believing in me, and your constant encouragement through even the most challenging times. Lastly to the Jung’s family, Steve, Stella, Christine, and Tiffani, this thesis would not be possible without your love and support.   1 Chapter 1:!Overview Vitamin B6 (B6) is an essential nutrient that plays crucial roles in the human body. The biologically active form of B6, pyridoxal-5’ phosphate (PLP), participates as a coenzyme in over 140 reactions ranging from the metabolism of amino acids, synthesis of neurotransmitters and heme, regulation of energy homeostasis to the one-carbon metabolism that is critical for methylation reactions and the nervous system. B6 deficiency is defined as having a plasma PLP concentration < 20 nmol/L independent of observable clinical signs or symptoms [2]. Suboptimal B6 status, defined as having a plasma PLP concentration between 20−30 nmol/L [3], has been associated with an increased risk of cardiovascular disease [4–9] and several types of cancer [10–14].  Adequate B6 status is crucial for women across all stages of life. Women of childbearing age should maintain an adequate B6 status since low preconceptional B6 status has been associated with an increased risk of preterm birth and early miscarriage [15], and a reduced probability of conception [16]. In addition, prenatal B6 supplementation has been linked with higher infant birth weight [17] and a lower incidence of preeclampsia [18]. A meta-analysis of five prospective cohort studies has shown that women with higher serum PLP concentration had a reduced risk of postmenopausal breast cancer [13]. Since cardiovascular disease and cancer are  2 also the top two causes of death in individuals aged >65 years in Canada [19], having an adequate B6 status is important for older adult women to have lifelong health.  According to the Canadian Community Health Survey (CCHS) in 2004 [20], 18% of Canadian adult women did not meet the Estimated Average Requirement (EAR; 1.1 mg/d) of B6, the value estimated to meet the dietary requirements of 50% of healthy individuals in a population. Specifically, a 19.4% prevalence of inadequate dietary B6 intake was reported for older adult women (51−70 years), compared to a 9.6% prevalence for young adult women (19−30 years). Yet, the CCHS lacks data on biochemical B6 status in Canadian women. The prevalence of B6 deficiency and suboptimal B6 status in Canadian women remains unclear.  Information on biochemical B6 status in Canadian women is so far limited. In the recent Alberta Pregnancy Outcomes and Nutrition cohort study, none of the 119 first-trimester pregnant women and only 0.2% of the 528 second-trimester pregnant women had B6 deficiency; the study did not report on suboptimal B6 status [21]. However, the high socioeconomic status of the sample and an over 94% prevalence of prenatal multivitamin supplement use likely explain the low prevalence. In the United States, B6 deficiency was reported in over 40% of young adult women (21−44 years) and in 24% of adults aged ≥65 years in the National Health and Nutrition Examination Survey (NHANES) 2003−2004 [22]. In light of these findings and the lack of data  3 on biochemical B6 status in Canada, there is an urgent need to investigate the B6 status of Canadian women. Oral contraceptive use [22,23], no supplement use [22–24], increased physical activity [24,25], smoking [22–25], lower socioeconomic status [24,25], non-Hispanic African ethnicity [22], and higher body mass index [22,24] have been associated with a decrease in B6 status in large-scale studies in different countries. However, there are no data available on predictors of B6 status in Canada. Understanding the relationship between B6 status and demographic and lifestyle characteristics will aid in the identification of vulnerable population groups for B6 deficiency and suboptimal B6 status and related health consequences in Canada.   My thesis projects were developed from the questions: what is the prevalence of B6 deficiency and suboptimal B6 status in young and older adult women in Metro Vancouver and what are the potential dietary, demographic and lifestyle related predictors associated with B6 deficiency and suboptimal B6 status in these different age groups?   4 Chapter 2:!Literature Review 2.1! Preamble In this review, I present current evidence highlighting the importance of and the need for research on vitamin B6 (B6) in Canadian adult women. This chapter starts with an introduction on the metabolic forms and functions of B6, followed by a thorough summary of the biomarkers for B6 status assessment including direct and functional indicators. I then present the food sources of B6 and recommendations for adequate B6 intake. The second part of this review describes the importance of having an adequate biochemical B6 status. Accordingly, I examine the dietary, demographic and lifestyle factors that have been associated with biochemical B6 status, define the categories of biochemical B6 status, and review the potential adverse health consequences of B6 deficiency and low B6 status, with an emphasis on women.  A high prevalence of B6 deficiency was reported in US women of childbearing age and older adult women. In Canada, 18% of adult women in a nationally representative sample had inadequate dietary B6 intake in 2004. To date, there is limited data on biochemical B6 status in Canadians. The last part of this review, I present the data available on dietary and biochemical B6 status in Canada, examine the current research gaps in Canada, and end with the rationale for investigating biochemical B6 status in Canadian women.  5 2.2! Metabolic Forms of Vitamin B6 Vitamin B6 (B6) is a micronutrient that is required for a wide range of metabolic processes. B6 is found in three interconvertible forms, also called B6 vitamers: pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM). These three vitamers can be phosphorylated into the following coenzymes: pyridoxine 5’-phosphate (PNP), pyridoxal 5’-phosphate (PLP), and pyridoxamine 5’-phosphate (PMP), which are important for many biological reactions. A glucoside derivative of PN also exists as pyridoxine 5’-β-D -glucoside (PNG). With the exception that PNG has only half of the bioavailability of other B6 vitamers in humans [26], PN, PM, PL and their phosphorylate forms (i.e. PNP, PLP and PMP) have similar bioavailability of 75% [27,28].  Vitamin B6 is water-soluble and is absorbed through passive diffusion in the small intestine in the form of PN, PL, or PM. Prior to absorption, PNP, PLP, and PMP require dephosphorylation by alkaline phosphatase and pyridoxal phosphate phosphatase [28,29]. The hydrolysis of PNG to PN partially occurs at the brush border membrane by the brush border lactase-phlorizin hydrolase [30,31]. PNG is also absorbed intact via passive diffusion and later hydrolyzed by the cytosolic PNG hydrolase in various tissues including liver and kidney [31–33]. Once absorbed, erythrocytes transport PL and PN in the hepatic portal vein along with free form PN, PL, PM and PNG to the liver [28]. After being absorbed in the liver, the non- 6 phosphorylated forms of B6 vitamers are phosphorylated and converted to PLP by pyridoxal phosphate kinase [2,34]. The liver releases PLP, and to a lesser extent also PL, both bound to albumin into the circulation [2,35] to be carried to tissues and organs for uptake and storage, mainly to skeleton muscles (accounting for 80% of total B6 in the body) [2,28]. Prior to uptake by tissue or transfer between cell compartments, alkaline phosphatase converts PLP to PL [36]. After diffusing into tissue cells, PL is phosphorylated to PLP by pyridoxal phosphate kinase, which is more stable and prevents subsequent diffusion out of the cells. The majority of PLP in the human body is found tightly bound to various proteins in the tissues to protect it from hydrolysis to its non-phosphorylate form and release as PL into circulation [2,34].  The B6 vitamers were described to have different levels of ‘bioactivity’; bioactivity is defined as ‘the biological activity (i.e. vitamin activity) of a vitamer tested relative to the appropriate reference form of that vitamin’ [27]. In the human body, PN is defined as the B6 vitamer with full B6 bioactivity, whereas PL and PM were reported to have slightly lower bioactivity in some studies [27,37]. The primary catabolite of all B6 vitamers in humans is 4-pyridoxic acid (4-PA). After B6 vitamers are catabolized in the liver, 4-PA is excreted in the urine. The conversion of B6 vitamers is summarized in Figure 2-1.  7   2.3! Functions of Vitamin B6 Vitamin B6 functions as a coenzyme in the form of PLP in over 140 reactions in the human body. Pyridoxal 5’-phosphate for example plays an essential role in the metabolism of amino acids where it assists in processes such as transamination, decarboxylation, racemization, aldol cleavage, and dehydration by forming a Schiff base or binding to an ε-amino group of a specific lysine residue [28,38,39]. In the central nervous system, PLP is involved in the synthesis of neurotransmitters such as serotonin, norepinephrine, epinephrine, and γ-aminobutyrate (GABA) [39]. Within energy metabolism, PLP regulates energy homeostasis through gluconeogenesis and glycogenolysis, as the coenzyme for glycogen phosphorylase. In hemoglobin synthesis, PLP participates as a coenzyme of 5-aminolevulinic acid synthase to form Figure 2-1 Conversion and Catabolism of Vitamin B6 Vitamers Abbreviations: PNPO, pyridoxamine 5′-phosphate oxidase; ALP, alkaline phosphatase; AT, aminotransferase; PDXP, pyridoxal phosphate phosphatase; PDXK, pyridoxal phosphate kinase; AOX, aldehyde oxidase(s). Adapted from [38].  8 heme, a component of hemoglobin, which affects the ability of erythrocytes to carry oxygen throughout the body. Furthermore, PLP has numerous coenzyme roles in the one-carbon metabolism that is critical for nucleotide formation, amino acid interconversion, and the formation of DNA, neurotransmitters, and phospholipids through transmethylation reactions. Pyridoxal 5’-phosphate is also required for the generation of methyl groups through the glycine cleavage system and the interconversion of glycine and serine, thereby facilitating methionine recycling and regulating homocysteine concentrations (Figure 2-2). In the transsulfuration pathway, PLP serves as a coenzyme for cystathionine β-synthase and cystathionine γ-lyase to convert homocysteine into cystathionine and cysteine [40]. In addition, PLP indirectly regulates the synthesis of nucleic acids by serving as a coenzyme for serine hydroxymethyltransferase (SHMT), which catalyzes the conversions of serine to glycine and tetrahydrofolate to 5,10-methylene tetrahydrofolate.   9   In tryptophan catabolism, PLP participates in the formation of nicotinamide adenine dinucleotide, the coenzyme form of niacin. The kynurenine pathway of tryptophan metabolism has two PLP-dependent enzymes: kynureninase and kynurenine transaminase (Figure 2-3) [34,38,41,42]. Figure 2-2 Roles of Vitamin B6 in One Carbon Metabolism  Abbreviations: BHMT, betaine hydroxymethyltransferase; B2, riboflavin; B12, cobalamin; CBS, cystathionine β-synthase; CGL, cystathionine γ-lyase; dTMP, deoxythymidine monophosphate; dUMP, deoxyurdine monophosphate; MAT, methionine adenyltransferase; Methylene THF, 5,10-methylene tetrahydrofolate; Methyl THF, 5-methyl tetrahydrofolate; MTHFR, 5,10-methylene tetrahydrofolate reductase; PLP, pyridoxal 5’-phosphate; SAH, S-adenosylhomocysteine; SAHH, S-adenosylhomocysteine hydrolase; SAM, S-adenosylmethionine; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; TYMS, thymidylate synthase. Adapted from [50,172,173]  10   2.4! Indicators for Vitamin B6 Status Assessment Biomarkers used for B6 status assessment can be categorized into direct and functional indicators, as summarized in Table 2-1. Direct indicators are circulating B6 vitamers; functional indicators are substrates of B6-dependent pathways and reflect the intracellular status of B6. 2.4.1! Direct Indicators of Vitamin B6 Status  Plasma PLP, PL, total B6 and 4-PA are direct indicators of B6 status [3,38]. Plasma PLP is the most commonly used indicator [3]. Animal models have demonstrated that plasma PLP Figure 2-3 Tryptophan Catabolism through Kynurenine Pathway  Abbreviations: PLP, pyridoxal 5’-phosphate; KYN, kynureninase; KAT, kynurenine transaminase. Adapted from [38]  11 concentration reflects PLP content in the liver, but not that in muscle tissue, which constitutes 70–80% of whole body B6 [28,38,43,44]. Plasma PLP responds to changes in dietary B6 intake, apparently plateauing 7–10 days after B6 repletion [3,45,46] and depletion [47,48]. Plasma PLP is the only indicator of B6 status that has an established cut-off for adequate and deficient status [2]. A plasma PLP concentration of 30 nmol/L has been suggested by Leklem [3] to reflect the lower end of adequate B6 status. However, results from many different studies with various population groups have shown that a substantial number of individuals (up to 50%) in these populations had plasma PLP concentrations below 30 nmol/L but without clinical data to suggest B6 deficiency. Thus, the Institute of Medicine has used a more conservative cut-off to define B6 adequacy, i.e., having a plasma PLP concentration > 20 nmol/L [2]. Having a plasma PLP concentration of 20–30 nmol/L has thereafter been referred to as suboptimal B6 status, also called marginal B6 deficiency [3]. However, it should be noted that plasma PLP is affected by factors other than dietary B6 intake, for example inflammation, albumin concentration, and alkaline phosphatase activity [38]. In light of these influencing factors, the validity of plasma PLP as B6 status indicator in diseased populations has been questioned, though it appears to be an appropriate indicator for B6 status assessment in healthy populations [38]. Other limitations of plasma PLP as blood biomarker are that PLP is sensitive to light and unstable at room temperature [38]. In conclusion, although plasma PLP is widely used as an indicator of B6 status,  12 it should be interpreted with caution in diseased population and in the case of inappropriate sample collection and storage. Although plasma PL has been identified as an additional direct measurement of B6 status, PL is unstable in many conditions such as in serum, heparin-treated plasma, and under light exposure; however it is stable in ethylenediaminetetraacetic acid (EDTA) treated plasma [3,38]. Total B6-aldehyde, defined as the total concentration of plasma PLP and PL, is considered relatively stable compared to PL alone and can also provide additional information about distribution and conversion of B6 vitamers in plasma samples [38]. Given that plasma B6-aldehyde is the total concentration of plasma PLP and PL, it accounts for the degradation of PLP into PL in plasma sample under room temperature and ultimately reflects approximately 90% of total B6 in plasma [3]. However, due to the lack of established reference ranges available for plasma PL or total B6, the applicability of these indicators in practice is lower compared to plasma PLP, for which established reference ranges and a cut-off for B6 deficiency exist.  Plasma 4-PA has also been recognized as an additional direct indicator of B6 status as it responds to changes in B6 intake within 1–2 weeks [3,40,49]. Plasma 4-PA is stable under room temperature and light exposures and is not affected by alkaline phosphatase activity [38], which simplifies the sample handling in comparison to the indicators PLP and PL. However, plasma 4-PA is highly influenced by renal function and therefore lacks specificity [38]. The ratio of 4-PA  13 to total B6 (PAr) has been validated as an indicator of increased B6 catabolism as it occurs during inflammation [38]. PAr has also been suggested as a predictor for the risk of lung cancer [14], a topic that will be discussed in section 2.8.4. 2.4.2! Functional Indicators of Vitamin B6 Status Functional indicators of B6 status can be divided into two categories: transaminase activity levels and circulating concentrations of metabolites from B6-dependent pathways. Transaminase activity tests measure the ratio of apo-enzyme to the total enzyme of PLP-dependent transaminases such as aspartic acid transaminase and alanine transaminase in erythrocytes. The activity levels of these transaminases reflect the long-term status of B6 because of the long lifespan (approximately 120 days) of erythrocytes [3]. Unlike plasma PLP or 4-PA, transaminase activity levels are not affected by albumin, alkaline phosphatase activity, immune indices, and kidney function; however, the activity is influenced by the presence of necrotic processes (e.g. myocardial infraction, muscle and liver damage) and alcohol consumption [38]. The biggest limitation of erythrocyte transaminase tests is that these tests need to be done within hours after the blood draw, which may not be practical in many research settings [38].  Metabolites that have been used as functional indicators for B6 status include plasma total homocysteine, cystathionine, and kynurenines. Due to the involvement of PLP as a coenzyme in the transsulfuration pathway, plasma total homocysteine has been used as a  14 functional indicator of B6. Plasma total homocysteine is commonly measured within clinical laboratories as part of standard diagnostic tests; however, plasma total homocysteine is more sensitive to folate and vitamin B12 status and thus a less specific indicator for B6 status [38,50].  Plasma cystathionine has recently been identified as a sensitive functional indicator owing to its prompt response to dietary B6 restriction in healthy adults and its negative correlation with circulating PLP [48,51,52]. Past studies have demonstrated that it is sensitive enough to detect suboptimal B6 status (plasma PLP concentration of 20–30 nmol/L) [48,51,52]. To date, it has not been studied whether circulating cystathionine concentrations differ between the type of collected biospecimen (e.g., heparin-treated plasma, EDTA treated plasma, and serum). It is also unclear whether cystathionine concentration is influenced by factors such as inflammation, enzyme activity, and body mass index (BMI). Future research is required to understand the variability and stability of cystathionine in plasma versus serum as a functional indicator of B6 status. Investigating the relation between cystathionine and other health indices will enhance the applicability of this biomarker. Plasma kynurenines, such as kynurenine, 3-hydroxykynurenine, xanthurenic acid, anthranilic acid, 3-hydroxyanthranilic acid, and kynurenic acid, have been evaluated as indicators of the B6-dependent tryptophan metabolism [38,41,42]. Plasma kynurenines are stable at room temperature but 3-hydroxykynurenine and 3-hydroxyanthranilic acid were found  15 unstable when plasma samples were placed on ice for 24 hours [38]. The ratio of 3-hydroxykynurenine to xanthurenic acid was proposed as a potential functional indicator of B6 status on the basis of its correlation with plasma PLP in a cohort of older adults with angina pectoris or aortic stenosis [41]. Although plasma 3-hydroxykynurenine was strongly correlated with inflammatory markers, kidney function, and circulating tryptophan [41], the ratios of 3-hydroxykynurenine to xanthurenic acid, of 3-hydroxykynurenine to 3-hydroxyanthranilic acid, and of 3-hydroxykynurenine to kynurenic acid have shown weaker or no associations with inflammation, BMI, and kidney function [38,53]. However, more data are required to derive reference ranges and validate the kynurenines as indicators of B6 status in both healthy and diseased populations. This research seems critical because plasma PLP as the single reference indicator for B6 status in diseased populations has its limitations.    16 Table 2-1 Summary of Vitamin B6 Status Indicators  Advantage Disadvantage Direct indicator Plasma PLP !!Cut-off for adequate/deficient status available [2] !!Light sensitive !!Unstable at room temperature !!Influenced by inflammation, albumin concentration, ALP activity, and alcohol consumption Plasma PL !!Stable in EDTA-treated plasma !!Light sensitive !!Unstable in heparin plasma sample at room temperature !!No reference values available Plasma total B6-aldehyde !!Summarized 90% of total B6 in plasma !!No reference values available Plasma 4-PA !!Stable under room temperature !!Not light sensitive !!Reflects short-term status !!Influenced by renal function !!No reference values available Functional indicator Erythrocyte aspartate (or alanine) transaminase activity !!Reflects long-term status !!Not related to albumin, ALP activity, immune indices, and kidney function !!Influenced by the presence of hemoglobin, necrotic processes, and alcohol consumption !!Requires the use of fresh blood Plasma total homocysteine •!Available in clinical laboratories as part of standard diagnostic indicators !!Influenced by folate and vitamin B12 status Plasma cystathionine !!Sensitive marker for suboptimal B6 status Determinants of cystathionine have not been investigated. Plasma kynurenines !!Plasma kynurenine ratios are not related to inflammation, BMI or kidney function. !!Unstable in plasma when placed on ice Abbreviation: 4-PA, 4-pyridoxic acid; ALP, alkaline phosphatase; B6, vitamin B6; BMI, body mass index; EDTA, ethylenediaminetetraacetic acid; PL, pyridoxal; PLP, pyridoxal 5’-phosphate.  17 2.5! Sources and Recommendation of Dietary Vitamin B6 2.5.1! Food Sources of Vitamin B6 Vitamin B6 is found in both plant and animal products. Based on the Canadian Nutrient File by Health Canada in 2015, food sources with high levels of B6 include meat, fish, poultry, liver, kidney, fortified cereals, soy products, nuts, and some vegetables and fruits [54]. In a cross-sectional study in an elderly Dutch population, meat, meat products, poultry, potatoes, and beverages (alcoholic and nonalcoholic drinks) were the major dietary sources of B6 [55]. In another cross-sectional study in European adolescents, B6 intake was associated with the intake of meat, starchy roots and potatoes, breakfast cereals, margarine, and oils [56]. There is no data on the main food sources of B6 in the multicultural Canadian diet. 2.5.2! Recommendations for Dietary Vitamin B6 Intake Dietary requirements provided by the Institute of Medicine were established using plasma PLP concentration ≥ 20 nmol/L as the main indicator of adequate B6 status, and using the tryptophan load tests or circulating 4-PA concentration in case plasma PLP concentrations were not available [2].  The estimated average requirement (EAR) for dietary B6 intake, which is the value estimated to meet the requirements of 50% of healthy individuals in a population, is 1.1 mg/day for both men and women aged 19–50 years [2]. The average requirement increases with age and  18 especially for men: the EAR for adults aged 50 years or older is 1.4 mg/day for men and 1.3 mg/day for women. The EAR is used to set a guideline for the mean intake of a specific population and to assess the prevalence of inadequate intake within a group [2].  By contrast, the recommended dietary allowance (RDA) is set as an intake guideline for individuals rather than a population [2]. The RDA for B6, which is the value estimated to meet the dietary requirements of 97–98% of the population, is set at 20% higher than that of the EAR [2] with the RDA for B6 as 1.3 mg/day for both men and women aged 19–50 years. For adults aged 50 years or older, the RDA increases to 1.7 mg/day for men and 1.5 mg/day for women [2]. Special considerations apply for estimating the dietary requirement of B6 for pregnant and lactating women. To compensate for increased metabolic needs and body weight during pregnancy, as well as the requirements of the fetus and placenta, the EAR and RDA for B6 for pregnant women are 1.6 and 1.9 mg/day, respectively [2]. For lactating women, the EAR and RDA for B6 are 1.7 and 2.0 mg/day, respectively, allowing for a sufficient concentration of B6 in human milk to facilitating an adequate B6 intake in breastfed infants [2]. The Dietary Reference Intake values for B6 are summarized in Table 2-2.     19 Table 2-2 Dietary Reference Intakes for Vitamin B6  Recommended Dietary Allowance (RDA), mg/day Estimated Average Requirement (EAR), mg/day Age Groups Men Women Men Women 19−30 years 1.3 1.3 1.1 1.1 31−50 years 1.3 1.3 1.1 1.1 51−70 years 1.7 1.5 1.4 1.3 70+ years 1.7 1.5 1.4 1.3 Pregnancy  1.9  1.6 Lactation  2.0  1.7 Adapted from [2].  2.6! Determinants and Predictors of Biochemical Vitamin B6 Status The following section summarizes factors that have been associated with B6 status: (1) dietary B6, (2) supplement use, (3) oral contraceptive and hormone therapy, (4) physical activity, (5) smoking, (6) socioeconomic status, (7) ethnicity, (8) alcohol consumption, and (9) body mass index (BMI). In this section, biochemical B6 status is mostly referred to plasma PLP concentration since PLP is the most commonly used indicator of B6 status and the only indicator with a set cut-off for adequate B6 status. In the studies reviewed in this section, B6 deficiency is defined as having a plasma PLP concentration < 20 nmol/L and low B6 status as having a plasma PLP concentration < 30 nmol/L. Other indicators such as plasma kynurenines are also discussed when applicable.  Information on the large-scale studies that are reviewed and frequently mentioned in this section are summarized in Table 2-3. 20 Table 2-3 Large-scale Studies Investigating Factors Affecting Vitamin B6 Status Reference Year and location Study design Sample size and age of participants Variables examined Main findings Deac et al. [23] 2015; Ireland (Dublin) Cross-sectional data in a cohort study n= 2,436;  18−28 years; male and female OC use, supplement use, smoking, alcohol consumption Metabolite concentrations in tryptophan metabolism differed between sex, supplemental B6 use, OC use, and alcohol consumption. Morris et al. [22] 2008; The United States (National) Cross-sectional survey (NHANES 2003−2004) n= 7,049;  ≥ 1 year; male and female Sex, age, B6 intake, OC, supplement use, ethnicity, smoking, BMI, alcohol consumption Smokers, non-Hispanic blacks, seniors, and current and former OC users were populations at risk for B6 deficiency. Pfeiffer et al. [24] 2013; The United States (National) Cross-sectional survey (NHANES 2003−2006) n= 4,489;  ≥ 20 years; male and female Age, sex, ethnicity, education, income, supplement use, smoking, alcohol consumption, BMI, physical activity Age, sex, income, supplement use, smoking, alcohol consumption, BMI, physical activity were significant predictors of plasma PLP concentration. Ye et al. [25] 2010; The United States (Boston) Cross-sectional data in a longitudinal study n= 1,236;  45−75 years; male and female Age, sex, education, income, smoking, alcohol consumption, physical activity High prevalence of low B6 status was found among individuals with household incomes below poverty, who were physically inactive, and current smokers Abbreviations: BMI, body mass index; NHANES, National Health and Nutrition Examination Survey; OC, oral contraceptive; PLP, pyridoxal 5’-phosphate  21 2.6.1! Dietary Vitamin B6 Dietary B6 can be found in foods such as meat, fish, poultry, liver, kidney, fortified cereals, soy products, nuts, and some vegetables and fruits [54]. Natural food sources of B6 contain different forms of B6 that partially differ in their bioavailability. Pyridoxal and PLP are the major forms in animal source foods while PN, PM, and their phosphorylated forms are the major forms of B6 present in plant foods [57]. Bioavailability is defined as the ‘fraction of an ingested nutrient that is available for utilization in normal physiologic functions and for storage’ [58]. Experiments conducted in pigs have revealed no significantly different bioavailability between PN, PL, and PM from different food sources [59]. However, the presence of PNG ranging from 5−75% of total B6 in plant products [60] have suggested a lower bioavailability of B6 from plant products compared to B6 from animal products. This is because PNG exhibits only about 50% bioavailability in humans [26,60] and it also shows weak antagonistic effects with PN [26]. In spite of this, vegetarian women did not have significantly lower plasma PLP concentration compared to non-vegetarian women when they had similar amount of dietary B6 intake [61]. The overall bioavailability of B6 from a mixed diet has been reported to be approximately 75% in healthy men by comparing the urinary B6 excretion with the excretion from the referent PN supplement [62]. According to the Institute of Medicine [2], every 1mg of B6 intake increases plasma PLP concentration by 19 nmol/L without adjusting for other variables (r= 0.90). In the report  22 by Morris et al. [22] on data from the US National Health and Nutrition Examination Survey (NHANES) 2003–2004, plasma PLP increased by 12 nmol/L per 1 mg increase in B6 intake from food and supplement sources, after adjustment for multiple demographic and lifestyle factors (n= 6,165; r= 0.32, P< 0.001). In contrast, a weak, yet statistically significant correlation was observed between plasma PLP and B6 intake from food and supplement sources (n= 1,130; r= 0.27, P< 0.001) and plasma PLP and B6 intake from food sources only (n= 767; r= 0.13, P= 0.002) after adjusting for other lifestyle and demographic variables in Puerto Rican adults aged 45−75 years living in Boston [25]. The reports from the Institute of Medicine [2] and Morris et al. [22] gave modest and similar estimations on the effect of B6 intake on plasma PLP. However, the weaker correlation coefficients between plasma PLP and B6 intake after adjustment for demographic and lifestyle factors from the study of Morris et al. [22] and Ye et al. [25] suggest that plasma PLP is also predicted by other variables such as smoking, income, and OC use in addition to B6 intake from food and supplement sources.  2.6.2! Supplement Use Studies have demonstrated that supplement use is an important predictor of B6 status. In the NHANES 2003–2004, Morris et al. [22] reported a total 11% of users of B6-containing supplements compared to 24% of non-users presenting with B6 deficiency (n= 6,159). Supplement use was positively associated with age, female sex, non-Hispanic white ethnicity, nonsmoking status, and BMI < 30 kg/m² [22]. However, among both supplemental B6 users  23 (n= 1,818) and non-users (n= 4,396), plasma PLP concentrations were significantly lower in women compared to men [22]. Deac et al. [23] also observed that users of B6-containing supplements (n= 671) had a 38% higher plasma PLP concentration than did non-users of supplements (n= 1,765; P< 0.001).  Furthermore, Pfeiffer et al. [24] examined the association between general supplement use (i.e., not only B6-containing supplements) and PLP concentration and reported an estimated 79% higher PLP concentration in supplement users (n= 2,271) compared with non-users (n= 2,215) after adjusting for multiple demographic and lifestyle variables [24]. The prevalence of B6 deficiency was 7.8% in supplement users and 19% in non-users [24]. The lower prevalence of B6 deficiency in supplement users might be explained by some B6-containing supplement users that were included in general supplement users. Supplement users have exhibited significantly higher plasma PLP concentrations and a lower prevalence of B6 deficiency compared with non-users of supplements in large-scale studies [22–24]. Therefore, supplement use should be considered as a variable when exploring the associations among B6 status and other predictors of B6 status.  2.6.3! Oral Contraceptive Use and Hormone Therapy The association between oral contraceptive (OC) and B6 status has been described since 1966 by Rose [63]. The use of OC has been reported to be inversely associated with plasma PLP concentration in both previous users and current users [22,23,64,65]. Morris et  24 al. [22] reported a high prevalence of B6 deficiency among women aged 21–44 years who did not take supplements containing B6. Among these women, the prevalence of B6 deficiency was 40% in former OC users (n= 227) and 78% in current users (n= 47) [22]. In the study by Deac et al. [23], lower plasma PLP concentration and abnormal concentrations of tryptophan metabolites were observed in OC users compared with nonusers, but the concentrations of other B6 vitamers (i.e. PL and 4-PA) did not significantly differ [23]. Studies have alluded to the possible mechanism linking OC use and low B6 status. Generally, studies proposed that although tryptophan metabolism is a B6-dependent pathway, the high content of estrogen in OC disrupts tryptophan metabolism independent of B6 status. In recent literature, the use of OC with lower doses of estrogen was also associated with low B6 status in a small sample size of 18 OC users and 11 non-users [66]. Hence, the mechanism behind low plasma PLP concentration and OC use remains unclear. The current hypothesis is that OC redistribute PLP between tissues rather than cause substantial insufficiency of B6 in the body [67]; whether this suggests a higher requirement of B6 intake for OC users is unclear and randomized controlled trials are required to ascertain this. Other hormonal contraceptives, such as the contraceptive patch, were not discussed in the studies mentioned above. Although the hormonal formula of the contraceptive patch and OC are both combinations of estrogen and progesterone, the route of application might modify the effects on metabolism and hemostasis of these hormonal contraceptives (transdermal versus oral)  25 [68].  Thus, more research is required to understand the effect of different types of hormonal contraceptives on B6 status.  Hormone replacement therapy that is usually provided to women in menopause is similarly inversely associated with plasma PLP as is OC use. Morris et al. [22] reported that among women aged > 44 years who were not taking B6-containing supplements, those reporting former or current hormone replacement therapy use had a slightly higher prevalence of B6 deficiency (27%; n= 275) compared to those not reporting hormone therapy use (23%; n= 157). In a double-blind, randomized controlled trial in 25 postmenopausal women with a screening plasma total homocysteine concentration >10 µmol/L, women who received a 13-week estrogen therapy (n= 8) or estrogen-progesterone therapy (n= 8) had significantly lower plasma PLP concentrations in comparison with the women in the placebo group (n= 9) [69]. The effect of hormone replacement therapy on plasma PLP in postmenopausal women seems relatively smaller than the association of OC use with plasma PLP concentration in young women. In Canada, the prevalence of OC use is 16.2% in women aged 15−49 years [70] and the prevalence of hormone therapy use is 15.3% in women aged 50−69 years [71]. Given the high prevalence of hormonal use, it is essential to incorporate this parameter in studies investigating B6 status in Canadian women.  26 2.6.4! Physical Activity Given that B6 participates in several metabolic processes that are highly active during exercise (specifically amino acid metabolism and glycogen breakdown), studies have suggested that physical activity increases B6 requirement [72,73]. However, this suggestion was mainly based on studies conducted in populations of professional athletes rather than the general population where physical activity levels vary [73,74]. Few studies have reported an association between physical activity and B6 status in the general population. In the NHANES 2005–2006, physical activity level was positively associated with plasma PLP concentration (n= 3,804; P< 0.001) after adjustment for demographic and lifestyle factors including supplement use, but not for dietary B6 intake  [24]. The highest mean plasma PLP concentration was observed in adults aged ≥ 20 years (n=1,176) who also reported the highest physical activity level [24]. However, the association between plasma PLP concentration and physical activity level was weak. Adults with a physical activity of 750 metabolic equivalent minutes per week had 3.1% higher plasma PLP concentration compared to those who had 150 metabolic equivalent minutes per week after adjusting for multiple demographic and lifestyle variables (n= 3,804) [24]. Similarly, Ye et al. reported that those categorized as physically inactive (defined as having a score < 30 of a physical activity questionnaire; n= 579) had significantly lower plasma PLP concentration and lower total B6 intake compared to those categorized as physically active (n= 656) [25]. In  27 contrast, a smaller cross-sectional study in 76 young adult men and women reported that neither plasma PLP concentration nor B6 intake differed between low and high physical activity levels [74]. The positive association between B6 status and physical activity levels has been identified in two large population-based studies [24,25] although inconsistent results were seen in the smaller study [74]. However, given the different assessment tools and categorizations for physical activity level between these studies, more research is needed to confirm that the association between physical activity and B6 status persist when using different assessment tools and categorizations. 2.6.5! Smoking Numerous studies identified significantly lower plasma PLP concentrations in smokers compared to non-smokers [22–24,75–77]. In the NHANES 2003−2004, current smokers had a significantly lower mean plasma PLP concentration and higher prevalence of B6 deficiency than never smokers did, independent of B6-containing supplement use [22]. Among non-users of B6-containing supplements, current smokers (n= 896) had a 22% lower mean plasma PLP concentration and 8% higher prevalence of B6 deficiency than never smokers (n= 3,411) [22]. Similarly, in the NHANES 2005−2006, Pfeiffer et al. reported that smoking was associated with a 28% lower plasma PLP concentration after adjusting for other demographic and lifestyle factors (n= 3,804) [24]. In a cross-sectional study of 1,191 older adults in England, Walmsley et al. [75] reported that current smokers compared to never  28 smokers and former smokers had significantly lower dietary B6 intake (assessed with 24-hour recalls) and had significantly lower plasma PLP concentrations after controlling for dietary B6 intake. In the study of Ye et al. [25], dietary B6 intake did not differ between current smokers (n= 298) and never smokers (n= 562) , but current smokers had significantly lower plasma PLP concentration and a higher prevalence of B6 deficiency and low B6 status compared to never smokers. Past studies have consistently shown lower plasma PLP concentration in current smokers; however, the association between dietary B6 intake and smoking was discrepant between different studies.  Results of the cross-sectional study with 1,191 older adults in England [75] suggested that the inverse relationship between smoking and plasma PLP is not attributable to dietary antioxidant or micronutrient intake but potentially to adverse physiologic consequences of smoking such as inflammation [75]. Similarly, another study proposed that smoking leads to an acute elevation in oxidative stress and engenders the redistribution of PLP in tissues [77]. This corresponds with the findings by Deac et al. [23], who reported a significant inverse association between smoking and plasma PLP concentration after adjusting for gender, OC use, B6 supplement use, and alcohol status (−16.2% lower plasma PLP concentration in non-smokers versus heavy smokers; P= 0.0007), but did not find an association between smoking and plasma concentrations of other B6 indicators such as plasma kynurenines, plasma PL or plasma 4-PA, in healthy young adults.   29 According to Statistics Canada, 18.1% of Canadians smoked in 2014 [78]. More specifically, 21.4% of males and 14.8% of females in Canada reported that they smoked [78]. This substantial prevalence of smoking emphasizes the importance of understanding whether the relationship of smoking and B6 status is causal. Including functional indicators such as plasma kynurenines to assess B6 status in future studies may help to elucidate the underlying mechanisms between smoking and B6 status. 2.6.6! Socioeconomic Status Socioeconomic status commonly refers to one’s combined economic and social status and is often measured as a combination of education, income, and occupation. Vitamin B6 status was reported to differ significantly between household income levels, but not between varying education levels [24,25]. Pfeiffer et al. observed significantly higher plasma PLP concentrations among individuals with higher income levels and this difference remained significant after adjusting for other demographic and lifestyle factors in the NHANES 2005–2006 (n= 3,804) [24]. A significantly higher plasma PLP concentration was also found in individuals of higher education level (n= 4,484) [24], but this difference attenuated after adjusting for other demographic and lifestyle factors. This finding suggests that the association between education level and PLP concentration was explained by other demographic and lifestyle factors. Similar results were reported by Ye et al. [25]; significantly lower plasma PLP concentration and a higher prevalence of both B6 deficiency and low B6  30 status were observed in Puerto Rican adults who lived in poverty (n= 360) compared with those who did not (n= 817). Moreover, Ye et al. [25] also did not find a significant difference in plasma PLP concentration between different education levels.  Low income households in Canada have a high susceptibility to food insecurity, defined as a household’s financial inability to access adequate food [79]. In the Canadian Community Health Survey (CCHS) 2004–2005, Kirkpatrick and Tarasuk identified significantly higher B6 intakes in persons without food insecurity compared with those who were experiencing food insecurity, across different age and sex groups except males aged 19−30 years and females aged 51−70 years [80]. This finding suggests that persons who have lower income levels potentially have lower B6 status because they are more likely to experience food insecurity. 2.6.7! Ethnicity Ethnicity was associated with B6 status in the NHANES 2003–2004 and the NHANES 2005–2006 [22,24]. In the NHANES 2003–2004, significantly lower plasma PLP concentration and higher prevalence of B6 deficiency were found in non-Hispanic blacks compared with non-Hispanic whites, independent of their B6-containing supplement use, after adjusting for protein and energy intake [22]. In the NHANES 2005–2006, a significant difference in plasma PLP concentration was found among non-Hispanic whites (n= 2,266), non-Hispanic blacks (n= 1,001), and Mexican-Americans (n= 912) [24]. However, there was  31 no significant association between plasma PLP and ethnicity after adjusting for demographic and lifestyle factors [24]. South Asians, i.e., people with ethnic origins from South Asian countries including Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan and Sri Lanka, are the largest ethnic minority group in Canada [81], but limited data is available on the nutritional status of this population. A previous study illustrated that acculturation changed the dietary pattern of 207 South Asian immigrants in Canada, who exhibited healthier food choices and food practices including an increased consumption of fruits and vegetables and an increase in grilling and a decrease in deep frying when cooking following immigration to Canada [82]. However, they also reported an increase in the consumption of convenience foods, sugar-sweetened beverages, red meat and in dining out [82]. In a cross-sectional study looking at cardiovascular disease risk in South Asian, Chinese, and European adolescents in Metro Toronto (n= 203), no significant difference in dietary macronutrient intakes was demonstrated between different ethnicities [83]. In a previous study from our research group [84], a non-significant yet higher prevalence of vitamin B12 deficiency was reported in 206 young adult women of South Asian ethnicity than in those of European ethnicity in Metro Vancouver. The difference was likely attributed to the low intake of animal source foods and lower socioeconomic status of South Asian women [84]. In contrast to vitamin B12, dietary sources of B6 are not limited to animal source foods [54]. Nevertheless, B6 status has been inversely  32 associated with socioeconomic status; thus, it is possible that South Asian women are more vulnerable to B6 deficiency than European women given that populations of South Asian ethnicity in Canada were reported to likely have incomes that fall below Statistics Canada’s low-income cutoffs and to very likely have low incomes than the overall Canadian population  [85]. To date, no study has examined B6 status in South Asian Canadians. On the basis that 25% of the total visible minority population and 4.8% of the Canadian population is of South Asian ethnicity [81], investigating B6 status in this population is imperative. 2.6.8! Alcohol Consumption Higher alcohol consumption has been associated with higher plasma PLP concentration [22,24,86]. Morris et al. [22] reported a tendency of higher mean plasma PLP concentration in alcohol consumers (n= 823) relative to non-consumers (n= 3,576) from the NHANES 2003−2004 (0.05≤ P ≤ 0.1). Alcohol consumption was positively associated with plasma PLP concentration in a cross-sectional study of 1,198 older adults aged ≥65 y in the UK (P= 0.03) [86]. Similarly, Pfeiffer et al. [24]  identified a positive correlation between alcohol consumption and plasma PLP concentration after adjusting for other demographic and lifestyle variables in the NHANES 2005–2006 (n= 3,804; P< 0.0001). Ye et al. [25] presented inconsistent results. In their study, current moderate alcohol consumers (≤1 drink/day for women or ≤ 2 drinks/day for men; n= 398) had a significantly higher plasma PLP concentration and lower prevalence of B6 deficiency compared to non-consumers (n=  33 746) [25]. However, current heavy alcohol consumers (defined as >1 drink/day for women and >2 drinks/day for men; n= 71) exhibited a slightly but not significantly higher mean plasma PLP concentration compared to non-consumers (n= 746) [25]. Dietary B6 intake did not differ between alcohol users and non-users in the study of Ye et al. [25]. Although alcohol consumption and dietary intake of B6 were positively associated in another study, among 10,286 current alcohol consumers (defined as ≥12 drinks in the entire life and had drinks at least one day in the past year) in the NHANES 1999–2006, dietary B6 intake was reported to significantly increase with alcohol consumption [87]. Most of the studies reported a positive association between B6 status and alcohol consumption [22,24,86] although one study found inconsistent results in heavy alcohol consumers [25]. Since moderate alcohol consumption has been reported to be associated with a reduced mortality, specifically a reduced risk of death from coronary heart disease in middle-aged and older adults [88], and because low B6 status has been associated with a higher risk of cardiovascular disease [4–9] (detailed in section 2.8.1), it would be important to understand the role of alcohol consumption in the relationship of B6 status and cardiovascular disease. 2.6.9! Body Mass Index   In the NHANES 2005–2006, BMI was negatively correlated with plasma PLP concentration (n= 4,417; r= –0.17, P< 0.05) [24]. Specifically, a 25% increase in BMI was associated with a 12.6% decrease in plasma PLP concentration after adjusting for  34 demographic and lifestyle variables (n= 3,804) [24]. However, inconsistent findings were reported when using BMI as a categorical variable (i.e., underweight, normal weight, overweight, obese). The mean plasma PLP concentration in individuals who were normal weight and overweight were very similar (56.7 versus 57.7 nmol/L; P-value not provided) [24]. In the NHANES 2003–2004, plasma PLP concentration did not differ between normal weight (BMI= 18.5–24.9 kg/m2; n= 1611) and overweight (BMI= 24.9–29.9 kg/m2; n= 1079) or obese (BMI ≥ 30 kg/m2; n= 1035) participants, but were significantly lower in underweight participants (BMI< 18.5 kg/m2; n= 674), in the sample of non-users of supplemental B6 [22]. In users of supplemental B6, plasma PLP concentrations were significantly lower in obese individuals (n= 397) and significantly higher in underweight individuals (n= 239) compared to PLP concentration of normal weight individuals (n= 583) (P< 0.05) [22]. In summary, the observation of an inverse relationship between plasma PLP concentration and BMI as a continuous variable was consistent between different large datasets in the US, but this relationship was not consistently observed when using BMI as a categorical variable.  The volumetric dilution of the blood was suggested as a potential underlying mechanism for the relationship between increasing body mass and decreasing concentrations of B6 and other B-vitamins [89]. In addition, plasma PLP concentration has been inversely  35 associated with the circulating concentration of C-reactive protein, a marker of systemic inflammation that is elevated in obesity [76,90]. 2.7! Cut-offs for Vitamin B6 Status and Health Consequences Associated with Vitamin B6 Deficiency and Suboptimal B6 Status 2.7.1! Categories of Vitamin B6 Status Limited scientific information is available concerning which concentration of a particular indicator represents B6 deficiency or low B6 status. As mentioned, the dietary reference intake values from the Institute of Medicine [2] were derived using plasma PLP concentration of < 20 nmol/L as the main indicator of B6 deficiency based on the study of Lui et al [91]. To note, this cut-off was not accompanied by any observable health risks but allowed a moderate safety margin to protect against the development of signs or symptoms of deficiency. Thereafter, several studies including the population-based NHANES in the US [22] defined B6 deficiency as having a plasma PLP concentration of < 20 nmol/L.  Suboptimal B6 status—also described as subclinical B6 deficiency or marginal B6 deficiency—is defined as having a plasma PLP concentration between 20–30 nmol/L [3]. In this thesis, low B6 status is defined as having plasma PLP concentration < 30nmol/L. Low B6 status has been linked with several chronic health conditions, including cardiovascular diseases and various types of cancers. Those associations are detailed in section 2.8.  36 In this thesis, I chose plasma PLP as the indicator of B6 status since it is the only indicator that has an established cut-off for adequate status [2]. B6 status is categorized as follows: plasma PLP > 30 nmol/L for adequate B6 status, < 30 nmol/L for low B6 status,  20–30 nmol/L for suboptimal B6 status [3], and < 20 nmol/L for B6 deficiency [2,3].  2.7.2! Clinical Consequences of Vitamin B6 Deficiency Clinical symptoms of severe B6 deficiency are rare and include microcytic anemia [92–95], convulsions, seizures [96,97], and peripheral neuropathy [98]. Microcytic anemia is caused by reduced synthesis of hemoglobin that occurs as PLP participates as a coenzyme in the metabolism of heme [93]. Because PLP is involved in the synthesis of GABA, chronic B6 deficiency can lead to a decrease in GABA concentrations in the brain, thereby increasing the risk for convulsions and seizures [99]. Because these clinical symptoms do not occur in all patients with B6 deficiency, they are not used to diagnose deficiency. B6 deficiency has also been associated with depression [100–102], confusion [100], and impaired immune responses [103,104] but more research is needed to clarify the direction of causality between B6 deficiency and these symptoms. 2.8! Health Implications Associated with Low Vitamin B6 Status 2.8.1! Cardiovascular Diseases  Low B6 status has been associated with cardiovascular disease [4–9]. In a population-based cohort study of 15,792 middle-aged adults, significantly lower plasma PLP  37 concentration (with no sex-specific difference) was observed in 232 cases of coronary heart disease compared to a reference cohort sample of 527 subjects [4]. In the same study, the risk of coronary heart disease was inversely associated with plasma PLP with the relative risk for the highest versus lowest quintile was 0.28 (95% CI = 0.1, 0.7) [4]. Similar findings were reported in a sample from a cohort study examining the B6 status and the risk of myocardial infarction in 32,826 women [5]. Among the 405 cases of myocardial infarction and controls, women with plasma PLP concentration ≥ 70 nmol/L exhibited 0.22 times the risk of myocardial infarction compared to those who had a plasma PLP < 27.9 nmol/L after adjusting for a list of demographic and lifestyle factors (95% CI = 0.09, 0.55) [5]. Another cohort study of 1,021 healthy men and women identified B6 status as a predictor of the risk of a cardiovascular event through a nested case-control study of 218 subjects [6]. The risk of a cardiovascular event decreased by 31% when plasma PLP concentration increased by 24.9 nmol/L (OR = 0.69; 95% CI = 0.49, 0.98) [6]. In summary, the inverse relationship between plasma PLP and the risk of cardiovascular disease has been consistently reported in different large-scale case-control studies. 2.8.2! Breast Cancer Breast cancer is the most common cancer among Canadian women: an estimated 1 in 9 females will develop breast cancer in their lifetime according to Canadian Cancer Statistics in 2016 [105]. Studies have identified an inverse association between plasma PLP and the  38 risk of breast cancer. A case-control study that was nested in a multi-ethnic cohort study in Hawaii and Los Angeles investigating the diet and cancer in the US identified 706 cases of breast cancer in 36,458 postmenopausal women aged 45−75 years at baseline [12]. Comparing these cases with 706 controls revealed that women with plasma PLP concentration > 116.6 nmol/L had a 30% lower risk of developing breast cancer than did women with plasma PLP concentration < 41.1 nmol/L (OR = 0.7; 95% CI = 0.50, 0.98) [12]. In addition, a meta-analysis of five prospective cohort studies reported a 20% lower risk of breast cancer in women who had serum PLP concentrations in the highest category compared to those with serum PLP concentrations in the lowest category (RR = 0.8; 95% CI = 0.66, 0.98) [13]. Furthermore, this association was significant in postmenopausal women, but not in premenopausal women (P < 0.001 versus 0.59) [13]. A dose–response relationship between serum PLP and the risk of breast cancer was also observed in this meta-analysis; for every 100 nmol/L increase in serum PLP concentration, the risk of breast cancer decreased by 23% (RR = 0.77; 95% CI = 0.69, 0.86) [13]. In summary, observational studies have reported an inverse association between PLP concentration and the risk of breast cancer, and this association was found to be more substantial in postmenopausal women. The reason for this strong, inverse association between B6 status and the risk of breast cancer in postmenopausal women is still unclear; however, it makes postmenopausal women a critical population to investigate for B6 status.  39 2.8.3! Colorectal Cancer   Colorectal cancer is the third and second most common type of cancer in Canadian women and men, respectively [105]. A meta-analysis of four prospective cohort studies determined that the risk of colorectal cancer in men and women with plasma PLP concentration in the highest category was 0.52 times that of adults with plasma PLP concentration in the lowest category (95% CI = 0.38, 0.71) [10]. Furthermore, the study identified a dose-response relationship between the risk of colorectal cancer and blood PLP concentration; a 100 nmol/L increase in blood PLP level reduced the risk of colorectal cancer by 49% (RR = 0.51; 95% CI = 0.38, 0.69) [10]. Similarly, among 223 cases and 407 controls in a multiethnic cohort study, people who had plasma PLP concentration > 101 nmol/L had 0.49 times higher odds ratio of developing colorectal cancer than did those with plasma PLP concentration < 26.6 nmol/L (95% CI = 0.29, 0.83) [11]. Another prospective case-control study also identified a 0.49 times relative risk of colorectal cancer in men with plasma PLP concentration > 144 nmol/L compared with those with a plasma PLP concentration < 43 nmol/L (95% CI = 0.26, 0.92) [106]. Studies have demonstrated a consistent and inverse association between plasma PLP and the risk of colorectal cancer, making the investigation of B6 status in Canadians critical while more research is needed to understand whether there is a causal relationship.  40 2.8.4! Lung Cancer  In Canada, lung cancer is the second and third most common cancer in women and men, respectively [105]. Studies on B6 status and lung cancer have presented inconsistent results. A case-control study of 899 lung cancer cases and 1,815 matched controls determined a 56% lower risk of lung cancer in individuals who had serum PLP concentration > 57.8 nmol/L compared to individuals who had serum PLP concentration < 28.4 nmol/L after adjusting for their smoking habits (OR = 0.44; 95% CI = 0.33, 0.60) [107]. In contrast, a prospective cohort study in 6539 Norwegian adults including 963 identified cancer cases after 11.9 years of follow-up [14], reported no association between the risk of lung cancer and plasma PLP but identified a relationship between lung cancer and PAr, which has been suggested as an indicator of B6 catabolism [14]. Every two standard deviations increase in PAr was associated with 2.46 times increased risk of lung cancer after adjusting for smoking and other lifestyle factors (95% CI = 1.49, 4.05) [14]. To summarize, studies have identified an inverse association between B6 status and the risk of lung cancer, but the findings are inconsistent. Future studies with a more comprehensive assessment of B6 status including direct and functional indicators may facilitate elucidating the relationship between B6 status and the risk of lung cancer.  41 2.8.5! Inflammation The development of cardiovascular disease and cancer has been related to inflammation in different studies [108–111]. Inflammation is involved in numerous mechanisms within these diseases, including the development and progression of atherosclerosis [110], proliferation and survival of cancer cells, angiogenesis, metastasis, subversion of adaptive immunity, and genetic instability [108]. Studies have proposed inflammation as the link between B6 status and cardiovascular disease and cancer [112,113], as mentioned in previous sub-sections. The association between low plasma PLP concentration and inflammation status is scientifically supported. In the NHANES 2003–2004, plasma PLP was reported to be inversely associated with the systematic inflammation marker C-reactive protein concentration, independent of B6 intake [76]. The odds ratios for an individual with a plasma PLP ≥ 20 nmol/L to have both moderately elevated and markedly elevated serum C-reactive protein concentrations were 0.52 (95%CI= 0.28, 0.96) and 0.17 (95% CI= 0.10, 0.31) after adjustment for all potentially confounding factors and B6 intake [76]. Moreover, a dose–response relationship between plasma PLP concentration and C-reactive protein concentration after adjustment of demographic and lifestyle factors including dietary B6 intake was observed in a cross-sectional study of 1205 Puerto Rican adults living in Boston [114]. A randomized controlled trial of patients with stable angina pectoris determined that B6 supplementation did not alter the inverse relationship between plasma  42 PLP and C-reactive protein [115]. The findings of these studies in disease populations suggest that low plasma PLP concentration is not attributable to B6 intake, but is potentially related to inflammation status. Most of the evidence indicated that inflammation causes the increased utilization of B6 in sites of inflammation, which leads to the redistribution of B6 and low circulating PLP concentrations, rather than that the low PLP concentration increases the risk of inflammation [112,113,116]. 2.9! Low Vitamin B6 Status in Women 2.9.1! Women of Childbearing Age Maintaining adequate B6 status is crucial for pregnant women and women of childbearing age. The increased requirement of dietary B6 in pregnant women compared to non-pregnant women is based on the increased body weight and metabolic requirements of the mother and the need of the fetus and placenta [2]. Plasma PLP concentration was shown to decrease throughout pregnancy, particularly during the third trimester [2,117]. However, whether this decrease indicates poor B6 status or is a normal physiological change is unclear [117–119].  Past research has shown that low maternal B6 status may compromise the woman’s health and increase the risk of poor pregnancy outcomes. A lower incidence of preeclampsia was reported in women supplementing with 10 mg/day of B6 during pregnancy compared with those who did not use supplements [18]. A lower Apgar score, an indicator of infant  43 birth health assessed by appearance, pulse, grimace, activity, and respiration within minutes of delivery, was rated in infants whose mother had lower maternal B6 status (erythrocyte alanine transaminase activity >25%) than infants whose mother had normal maternal B6 status [120]. Lower mean serum B6 concentration was found in mothers whose infant had an Apgar score <7 compared to mothers whose infants had an Apgar score ≥7 [121]. In a prospective cohort study of 458 young Chinese women, women who experienced an early miscarriage had significantly lower preconceptional plasma PLP concentration than women with a healthy pregnancy outcome [15]. Also, inadequate preconceptional B6 status has been associated with a reduced probability of conception [16]. On the contrary, in a meta-analysis of maternal B6 intervention studies (n= 247), maternal B6 supplementation (2.6 to ≥50 mg/day) was associated with a 217 g increase in infant birth weight [122]. Furthermore, high prevalence of low B6 status was also reported in Chinese women of childbearing age [123]; 26% of women had low B6 status and 39% had a combined deficiency of folate and B6 [123]. Many health consequences of dietary inadequacies before or at conception cannot or not fully be remedied as embryonic growth involves irreversible critical stages. Considering that an estimated 50% of pregnancies are unplanned [124], it is essential for women to maintain an adequate B6 status throughout their reproductive years.   44 2.9.2! Older Adult Women In 2015, Statistics Canada reported that the number of individuals in the population who were aged ≥65 years has increased to a new high of 16.1% being the first time exceeding the population of children aged 0−14 years [125]. Cardiovascular diseases and cancer are the top two causes of death in people aged >65 years in both Canada and the US, according to Statistics Canada in 2012 [19] and the US Center for Disease Control in 2015 [126]. Low B6 status has been associated with many chronic conditions, including cardiovascular diseases and several types of cancer, as reviewed in Chapter 2.9. Moreover, lower B6 status has also been associated with an increased risk of cognitive decline in a 4-year follow-up study of healthy older adults in Ireland (n= 662) [127]. Plasma PLP concentration <43 nmol/L was associated with a 3.5 times higher risk of accelerated cognitive decline, after adjustment for age and baseline mental state score (OR, 3.48; 95% CI= 1.58, 7.63) [128]. Thus, it is critical to understand the B6 status and the prevalence of low B6 status and B6 deficiency in the senior population. In the study of Ye et al. [25], Puerto Rican women aged 45−75 years had a 10.7% of B6 deficiency and a 17.8% of suboptimal B6 status. In the NHANES 2003–2004, B6 deficiency was detected in 24% of adults aged >65 years who were non-users of B6-containing supplements [22]. The prevalence of B6 deficiency was 32% in women and 16% in men who were not using B6-containing supplements, which suggests that women are more  45 vulnerable to low B6 status [22].  In a study in Norway, a 49% prevalence of B6 deficiency was observed in 60 nursing home residents who had a mean age of 85.3 years and of whom 61% were at risk of malnutrition [129]. Low serum PLP concentration of a median of 25.2 nmol/L was reported among older adults in Italy (n= 1320; median age= 69 years) [130]. Moreover, ever 10 years of age was significantly associated with a 3.5% decrease (standard error= 0.6; P < 0.001) in serum PLP concentration after adjustment of inflammation markers, dietary intakes, vitamin B12 status, and folate status [130]. These studies suggest that a high percentage of older adults have low B6 status, which may not relate to inflammation status or dietary intake. Given that low B6 status has been associated with many chronic conditions, more research is required to understand the causes and consequences of low B6 status in older adults.  2.10! Vitamin B6 Status of Canadians The B6 status of women in Canada remains unclear because large-scale, representative data on biochemical B6 status among Canadians are lacking. Moreover, few local studies have investigated biochemical B6 status in Canadian women. In the recent Alberta Pregnancy Outcomes and Nutrition cohort study, none of the 119 pregnant women in the first trimester and only 1 of 528 (0.2%) second-trimester pregnant women were classified as being B6 deficient [21]. The prevalence of suboptimal B6 status was not reported [21]; however, the high socioeconomic status and a greater than 94% prevalence of prenatal  46 multivitamin supplement use provided a homogenous study population with very little B6 deficiency [21]. A cross-sectional study comparing B6 status in 30 pre- and 30 postmenopausal women in Moncton, New Brunswick, showed that the mean plasma PLP concentration in pre- and postmenopausal women reflected adequate B6 status [131]. Nevertheless, these results should be interpreted with caution because of the non-representative sample size of 30 women in each group [131]. The only and latest national data on B6 status in Canadians is information about the dietary B6 intake in Canadians aged ≥1 years from the Canadian Community Health Survey (CCHS) in 2004 [20]. While the mean dietary B6 intake of Canadian adult women were above the age-specific EAR in all age groups, 17.7% of Canadian adult women did not meet the EAR of B6 from dietary sources  [20]. This prevalence of inadequate dietary intake increased from 9.6% in women aged 19−30 years to 32.5% in women aged >70 years (Table 2!4). These data indicate that a substantial percentage of Canadian women are at risk of B6 deficiency, especially with increasing age. In light of these findings and the high prevalence of B6 deficiency in US women [22], a similar prevalence of B6 deficiency is hypothesized in Canadian women.     47 Table 2-4 Usual Dietary Vitamin B6 Intake from Food Sources in Canadian Adult Women, Canadian Community Household Survey 2004 Age, years Sample size, n EAR, mg/day  Mean ± Standard Error, mg/day  <EAR, % 19−30 1854 1.1 1.59 ± 0.04 9.6 31−50 2686 1.1 1.65 ± 0.03 15.9 51−70 3200 1.3 1.66 ± 0.03 19.4 >70 2610 1.3 1.55 ± 0.03 32.5 19+ 10350  1.63 ± 0.02 17.7 Adapted from Health Canada [20].  2.11! Summary and Rationale Vitamin B6, in form of PLP, serves as a coenzyme for over 140 reactions.  PLP has a fundamental role in the metabolism of amino acids, synthesis of neurotransmitter and heme, regulation of energy homeostasis, and one-carbon metabolism. Maternal B6 adequacy is crucial at conception and throughout pregnancy to ensure healthy pregnancy outcomes. Due to an estimated 50% of pregnancies being unplanned, it is critical for women to maintain an adequate B6 status throughout reproductive years. Further, low B6 status has been associated with an increased risk of cardiovascular diseases and several types of cancer. Given that the leading causes of death in Canadians are cancer and cardiovascular diseases, further research focusing on the older, ageing population is essential. With women being at a greater risk for B6 deficiency, older women should be the focus when designing new studies.  Despite the essential role of B6 in health, no nationally representative data are available on the biochemical B6 status of Canadian women. Data from the CCHS in 2004 revealed an 18% prevalence of dietary B6 inadequacy in Canadian adult women; however, whether the dietary inadequacy is linked with biochemical B6 deficiency has not been  48 investigated. A high prevalence of B6 deficiency in adults, especially women of childbearing age and older adult women, was reported in the US. These findings necessitate the urge to investigate the B6 status of Canadian women. In several other countries (e.g., US, Ireland, and Norway), various socioeconomic and lifestyle factors have been associated with plasma PLP concentrations, including OC use, supplement use, smoking, physical inactivity, socioeconomic status, alcohol consumption, and BMI. Determining predictors of low B6 status in the Canadian population can contribute to the identification of vulnerable populations groups for low B6 status and related health consequences in Canada. This thesis will investigate the prevalence of suboptimal B6 status and B6 deficiency in women of childbearing age and older adult women in Metro Vancouver using plasma PLP concentration, the most commonly used and direct indicator of B6 status, and other functional biomarkers of B6 status. Demographic, dietary, and lifestyle predictors associated with plasma PLP will also be assessed.   49 Chapter 3:!Prevalence and Predictors of Low Vitamin B6 Status in Healthy Young Adult Women in Metro Vancouver 3.1! Introduction The overall objective of the research described in this chapter was to assess the prevalence of vitamin B6 (B6) deficiency and predictors of B6 status in a convenience sample of young adult women in Metro Vancouver.  The primary objective was to determine the biochemical B6 status and the prevalence of B6 deficiency and suboptimal B6 status in young adult women aged 19−35 years in Metro Vancouver, using plasma PLP as the primary status indicator. A high prevalence of B6 deficiency was reported among young adult women in the US National Health and Nutrition Survey (NHANES) [22]. However, there are no nationally representative data on the biochemical B6 status of Canadian women. The goal is to provide data on the prevalence of B6 deficiency in Canadian young adult women.  Various socioeconomic and lifestyle factors have been associated with plasma PLP concentrations in large-scale studies. The secondary objective was to determine demographic, dietary, and lifestyle predictors associated with plasma PLP concentration in this sample of young adult women. Finding predictors of low B6 status in a Canadian population will contribute to the identification of vulnerable populations groups for low B6 status and related health consequences in Canada.  50 3.2! Methods 3.2.1! Study Design and Participants  This study used data from a descriptive cross-sectional study conducted between 2012 and 2013 [84]. The recruitment and methods of the original study have been described in detail by Teo Quay in sections 3.3–3.5 of [132] and also in [84]. A total of 207 non-pregnant women aged 19−35 years of either South Asian or European ethnicity living in Metro Vancouver were recruited by convenience sampling. Eligibility of the potential research participants was determined by using a screening questionnaire; in short, participants must be aged between 19−35 years, have at least 3 grandparents from a single ethnic background of either European or South Asian (including Bangladeshi, Bengali, East Indian, Goan, Gujarati, Hindu, Ismaili, Kashmiri, Nepali, Pakistani, Punjabi, Sikh, Sinhalese, Sri Lankan, and Tamil ethnic groups) , and currently reside in Metro Vancouver. Participants must not suffer from any chronic condition for which they are receiving treatment or medication, currently take certain prescription medications, performance enhancing or recreational drugs, or over the counter medications, be pregnant, breastfeeding or lactating, or suffer from an active gastrointestinal condition or have undergone gastric bypass surgery. Informed written consent was obtained from all participants in the study.  51 Human ethics approval was granted by the UBC Clinical Research Ethics Board (#H11-01216), Vancouver Coastal Health Research Ethics Board (#V11-01216), and Fraser Health Research Ethics Board (#FHREB 2013-016). 3.2.2! Biospecimen and Data Collection Fasting venous blood samples were obtained from participants during a single clinic visit [84,132]. Samples were collected into lithium heparin vacutainers. Plasma was separated using centrifugation and stored at −80°C until analyses. The questionnaires including a demographic questionnaire, the “International Physical Activity Questionnaire (IPAQ) – Short Last 7 Day Self-Administered Format” [133,134], and a semi-quantitative Food Frequency Questionnaire (FFQ) “Your dietary intake” [135], were self-administered under the supervision of research staff during the clinic visit. Anthropometric measurements, including height, weight and waist circumference, were taken by research staff during the clinic visit. BMI was calculated based on weight and height (i.e., weight (kg)/ height(m)^2). The demographic questionnaire collected information on age, ethnicity, immigration, location, education, total household income, lactose intolerance, pregnancy history, oral contraceptive (OC) use, and supplement use. The IPAQ asked the participants to recall their physical activities in the past seven days categorized as walking, moderate-intensity activities, and vigorous intensity activities. Each activity was recorded with its frequency (days per week) and duration (time per day). Participants were categorized into three physical  52 activity levels, i.e., low, medium and high, according to the IPAQ analysis protocol [136]. The FFQ asked the frequency and portion of 78 food items, food practices, and lifestyle and activity questions in the past year. The database for calculating nutrient intakes used the information from the Canadian Nutrient File (2007b, Health Canada, 1982). The criteria used to detect implausibility included full blank page(s), more than 10% food items had missing answers for frequency or portion, or if energy intakes were <800 kcal/day or >4000 kcal/day [132]. However, none of the questionnaires collected met the criteria for outliers. Eighteen questionnaires were excluded because of high intake values >3 standard deviations (SD) from the mean intake of four Canada’s Food Guide food groups (grains, dairy, fruits and vegetables, and meat and alternatives), total energy, total protein, animal protein, and vitamin B12 [132]. 3.2.3! Laboratory Analysis Blood samples of 202 participants were available for the analysis of plasma PLP. Plasma PLP was measured by quantification of its semicarbazide derivative using high-performance liquid chromatography (HPLC) with fluorescence detection [137]. Briefly,  250 µL of plasma was mixed with 250 µL deionized water and 500 µL 10% trichloroacetic acid and centrifuged (10,000 rpm at 4°C for 5 min); 750 µL supernatant was then mixed with 50 µL 0.5 M semicarbazide and incubated in 37°C water bath for 15 min. After cooling for 15 min, the sample underwent liquid-liquid-extraction twice with 3 mL ethyl ether. Two  53 hundred µL of the clean extract was plated and 40µL was injected. Chromatography was carried out using an Agilent® 1260 HPLC with a reversed phase C-18 column (4.6x100 mm, particle size 3.5 µm; Agilent® Zorbax Eclipse Plus) and C-18 guard column (Agilent® Zorbax Eclipse Plus). PLP elution was achieved with a mobile phase of 97% 0.05M phosphate buffer and 3% acetonitrile at a flow rate of 1.1 mL/min followed by a wash step. Run time was 10 minutes per sample. PLP concentrations were quantified by external standardization method using peak areas. Agilent® software (Version 1.5.2) was used for HPLC control, data acquisition, and data processing. The assay showed good agreement and high precision when measuring external controls at low, medium, and high PLP concentrations, with intra- and inter-assay coefficients of variation of 2.6%, 2.2%, and 2.3%, and 15.7%, 9.5%, and 10.2%, respectively. The cut-off values used for B6 status in this thesis are as follows: plasma PLP concentration < 20 nmol/L for B6 deficiency [2,3], plasma PLP = 20–30 nmol/L for suboptimal B6 status [3], plasma PLP ≤ 30 nmol/L for low B6 status (i.e., combined B6 deficiency and suboptimal B6 status), and plasma PLP > 30 nmol/L for B6 adequacy. 3.2.4! Final Variables Included in the Analysis  Final variables from all questionnaires and biological assessment included in the analysis are summarized in Table 3-1.   54 Because of the low number of participants (n= 8) indicating secondary school education or less than secondary school education, education was dichotomized using the following cut-offs: low education, i.e., education below Bachelor’s degree, and high education, i.e., equal or higher than Bachelor’s degree.  Due to the low number of participants (n= 36) in the lowest household income bracket, household income was dichotomized into the following categories: low income, i.e., total annual household income <$30000 if 1−2 people, <$40000 if 3−4 people, <$60000 if ≥ 5 people, and high income, i.e., total annual household income ≥$30000 if 1−2 people, ≥$40000 if 3−4 people, ≥$60000 if ≥ 5 people.  Participants who reported current consumption of any nutritional vitamin or mineral supplements were classified as nutritional supplement users. Participants were asked to bring in nutritional supplement bottles. Brand names and frequencies of intake were recorded and B6 dosages were obtained retrospectively from web-based product information. Participants who were currently taking supplements containing B6 were classified as supplemental B6 users.  Participants were asked whether they were a: non-smoker, former smoker, current occasional smoker (1−9 cigarettes/d), current regular smoker (10−19 cigarettes/d) and current frequent smoker (≥ 20 cigarettes/d). Due to the high number of non-smokers (n= 174),  55 smoking data were dichotomized to non-smoker and smoker (including current smokers and former smokers).   Table 3-1 Variables and Coding Method Included in the Analysis of Vitamin B6 Status in Young Adult Women in Metro Vancouver Variable Type Unit or coding Height Continuous cm Weight Continuous kg Age Continuous years Waist circumference Continuous cm Body mass index Continuous kg/m² Ethnicity Categorical 1=European, 2=South Asian Physical activity Categorical 1=Low, 2=Medium, 3=High Oral Contraceptives Categorical 0=No, 1=Former, 2=Current Nutritional supplement  Categorical 0=No, 1=Yes Supplemental B6 use Categorical 0=No, 1=Yes Smoker Categorical I. 0=Non-smoker, 1=Former smoker, 2=Current occasional smoker (1–9 cigarettes/day), 3=Current regular smoker (10–19 cigarettes/day), 4=current frequent smoker (≥20 cigarettes/day)  Categorical II. 0=Never, 1=Former, 2=Current Income Categorical I. 1=Low, 2=Low-middle, 3=Middle-high, 4=High  Categorical II.4 0=High, 1=Low Education Categorical I.  1=Less than secondary school education, 2=Secondary school diploma, 3=Post-secondary education, 4=Bachelor’s degree, 5=University degree or >than bachelor’s degree  Categorical II.5 0=High, 1=Low Anemia Categorical Hemoglobin ≥12=No, hemoglobin <12=Yes Plasma PLP Continuous nmol/L  Categorical > 30=adequate status, 20-30=suboptimal status, < 20=deficiency Energy intake Continuous kcal Protein intake Continuous g Dietary B6 intake Continuous mg Adapted from [132]                                                    4"Low"income:"total"annual"household"income"<$30000"if"1−2"people,"<$40000"if"3−4"people,"<$60000"if"≥"5"people,"and"high"income:"total"annual"household"income"≥$30000"if"1−2"people,"≥$40000"if"3−4"people,"≥$60000"if"≥"5"people"5"Low"education:"education"below"Bachelor’s"degree,"and"high"education:"equal"or"higher"than"Bachelor’s"degree" 56 3.2.5! Statistical Analyses All statistical analyses were performed using R software (3.1.2 windows version). 3.2.5.1! Preliminary Univariate Analysis  Variables were described depending on the level of measurement. Continuous variables were examined for normality by visual histogram assessment. Normally distributed variables were reported as the arithmetic mean ± SD. Non-normally distributed variables were log-transformed and reported as geometric mean (95% CI). Categorical variables were presented by a frequency table. Statistical significance was set at a two-sided p-value of < 0.05. 3.2.5.2! Primary Objective The primary objective was to determine the biochemical B6 status and the prevalence of B6 deficiency and suboptimal B6 status in young adult women in Metro Vancouver. The primary outcome of this study, B6 status, was described using plasma PLP and the cut-offs for adequate B6 status, suboptimal B6 status, and B6 deficiency, as described above. Plasma PLP concentration was log-transformed to carry out the statistical analyses and was presented as geometric mean (95% CI). The prevalence of B6 deficiency and suboptimal status was presented as frequency (%).  57 3.2.5.3! Secondary Objective  The secondary objective was to identify demographic, dietary, and lifestyle predictors associated with B6 status, using plasma PLP as the status indicator. To identify the predictors of B6 status, multivariable linear regression was created with plasma PLP concentration as the dependent variable. For example:  Multiple linear regression model e.g. Plasma&PLP&concentration = &01 &+&03 46&6789:; +&0< 4=> +&0? @A; +⋯ The method for multivariable model construction is presented below: Bivariate analysis was conducted to identify variables associated with plasma PLP. If the p-value from the bivariate analysis was ≤0.2, the variable was included in the full model. A two-sample t-test was used for dichotomous variables, including ethnicity, education level, total household income level, OC use, supplemental B6 use, and smoking. One-way ANOVA followed by Tukey’s Honest Significance test was used for categorical variables with more than two levels including physical activity level. Simple linear regression was used for continuous variables, including dietary B6 intake, protein intake, BMI, and age. The association of the categorical variable B6 status, using plasma PLP cutoffs for adequate B6 status, suboptimal B6 status, and B6 deficiency, with potentially influential categorical variables, including ethnicity, education level, total household income level, OC use, supplemental B6 use, smoking and physical activity level, was tested using a Chi-square test.  58 All analyses as described above were also conducted after stratification for supplemental B6 use. Confounding and collinearity were checked by dropping one variable each time and compared the changes of effect size (03,&0<…) and the standard errors of the effect size (CD 3,&CD<…). A confounder was identified as the effect size of any other variables changes > 10%. A collinearity was identified as the standard error of the effect size of any other variables changes > 10%. Backward elimination procedure was used to establish the best-fit multivariable regression model. Partial F test or Akaike’s Information Criterion was used to select the best model, depending on the types of models that were being compared.   3.3! Results 3.3.1! Participant Characteristics Demographic, anthropometric, and lifestyle characteristics of the participants are summarized in Table 3-2. Most participants in the sample were of European ethnicity  (n= 147; 73%). The age of the participants was between 19 to 35 years old. Nineteen percent (n= 38) of participants were overweight6 and 8% (n= 16) obese. Overall, participants were                                                    6"South"Asian"cutHoffs"<18.5"(underweight),"18.6H23.9"(normal),"24H26.9"(overweight),">27"(obese);"European"cutHoffs"<18.5"(underweight),"18.6H24.9"(normal),"25H29.9"(overweight),">30"(obese)" 59 highly educated, with 71% (n= 144) having a Bachelor’s degree or higher. Of the 183 participants who provided information on their total household income status, 56 % (n= 103) were in the upper-middle or highest bracket for total household income. Based on the education and income indicators, the sample was of higher socioeconomic status.  Table 3-2 Demographic, Anthropometric, and Lifestyle Characteristics in Young Adult Women in Metro Vancouver Variables Distribution Total (n=202) Age*, years Normal 26.7 ± 4.2 Weight†, kg Skewed 62.7 (61.3, 64.2) Height*, cm Normal 166.4 ± 6.8 Body mass index†, kg/m² Skewed 22.7 (22.2, 23.1) Waist circumference, cm  Skewed 74.9 (73.7, 76.1) Ethnicity‡   European  147 (73) South Asian  55 (27) Education‡   Less than secondary school education  2 (1) Secondary school diploma  5 (3) Post-secondary education  51 (25) Bachelor’s degree  87 (43) University degree or >than bachelor’s degree  57 (28) Total household income‡§   Lowest  36 (20) Lower-middle  44 (24) Upper-middle  52 (28) Highest  51 (28) * Values presented as arithmetic mean ± SD.  † Values presented as geometric mean (95% CI). ‡#Values presented as n (%). §Lowest: <$15000 if 1−2 people, <$20000 if 3−4 people, <$30000 if ≥5 people, Lower-middle: $15000−29999 if 1−2 people, $20000−39999 if 3−4 people, $30000−50000 if ≥5 people, Upper-middle: $30000−59999 if 1−2 people, $40000−79999 if 3−4 people, $60000−79999 if ≥5 people, Highest: >$60000 if 1−2 people, >$80000 if ≥3 people.  Lifestyle characteristics of the participants are summarized in Table 3-3. Of the 200 participants who completed the physical activity questionnaire, 41% (n= 82) had a high level of physical activity. The use of OC was not common as 29% (n= 58) of the participants  60 reported the use of OC. About half of the participants (n= 97; 48%) were using nutritional supplements but 28% (n= 56) of the participants were using supplements that contain B6. As mentioned, the number of smokers was very small: only 5% (n= 12) were current smokers and 8% (n= 16) were former smokers.   Table 3-3 Lifestyle Characteristics in Young Adult Women in Metro Vancouver Variables Total (n=202) Physical activity level†  Low 8 (4) Medium 110 (55) High 82 (41) Use of oral contraceptives  No 144 (71) Yes 58 (29) Use of nutritional supplements  No 105 (52) Yes 97 (48) Use of supplemental vitamin B6  No 146 (72) Yes 56 (28) Current or former smoker  Never 174 (86) Former smoker 16 (8) Current occasional smoker (1–9 cigarettes/day) 7 (3) Current regular smoker (10–19 cigarettes/day) 4 (2) Current frequent smoker (≥20 cigarettes/day) 1 (0) *Values presented as n (%). †#Only 200 participants completed physical activity questionnaire.  3.3.2! Dietary Intakes and Associations with Food Sources Although all women completed the FFQ, only data from 184 participants were included in the dietary analysis due to 18 implausible reports of the FFQ. The mean total energy intake was 1566 (95% CI= 1504− 1629) kcal/day (Table 3−4). Given that the FFQ was semi-quantitative, dietary B6 intake was presented as relative dietary B6 intake.  61 Quartiles of dietary B6 intake were 0–1.1 mg/day, 1.1–1.4 mg/day, 1.4–1.7 mg/day and >1.7 mg/day, respectively.  Table 3-4Dietary Intake in Young Adult Women in Metro Vancouver Variables Distribution Total (n=184) Total energy intake*, kcal/day Skewed 1566 (1504, 1629) Protein intake†, g/day Normal 63.1 ± 21.1 Relative dietary vitamin B6 intake, mg/day Skewed  Q1  <1.1 Q2  1.1–1.4 Q3  1.4–1.7 Q4  >1.7 * Values presented as geometric mean (95% CI). †Values presented as arithmetic mean ± SD.   The following multivariable analysis was done to explore the four food groups defined in Canada’s Food Guide that contributes the most to the dietary B6 intake. All four food categories significantly contribute to dietary B6 intake, as shown by multiple linear regression of dietary B6 intake in Table 3-5. However, dietary B6 intake was derived mainly from meat and meat alternatives. One serving of fruits and vegetables or dairy provided only about half of the B6 intake that a serving of meat and meat alternatives provided.  Table 3-5Food Sources of Vitamin B6 in Young Adult Women in Metro Vancouver Variables Adjusted change in dietary B6 intake, mg/day (95%CI) Adjusted P-value Grains, serving 0.03 (0.01, 0.04) <0.001 Fruits and Vegetables, serving 0.10 (0.08, 0.11) <0.001 Dairy, serving 0.10 (0.07, 0.13) <0.001 Meat and Meat Alternatives, serving 0.19 (0.16, 0.22) <0.001 Adjusted changes and p-value were from multiple linear regression model controlled for intake of grains, fruits and vegetables, dairy, and meat and alternatives. Number of observation = 184, Model P < 0.001, R²: 0.79, Adjusted R²: 0.79. Abbreviations: B6, vitamin B6  62 3.3.3! Primary Objective: Prevalence of Vitamin B6 Deficiency and Suboptimal Vitamin B6 Status  The distribution of plasma PLP concentrations was skewed to the left (Figure 3-1) and thus log-transformed to perform all further analyses. Mean (95% CI) concentration of plasma PLP was 61.0 (55.2, 67.3) nmol/L (Table 3-6). The prevalence of B6 deficiency was 1.5% and that of suboptimal B6 status was 10.9%.     Figure 3-1 Distribution of Plasma Pyridoxal 5’-phosphate Concentration in Young Adult Women in Metro Vancouver  63 Table 3-6 Plasma Pyridoxal 5’-phosphate Concentration and Prevalence of Vitamin B6 Deficiency, Suboptimal Status, and Adequacy in Young Adult Women in Metro Vancouver Variables Distribution Total (n=202) Plasma PLP concentration†, nmol/L Skewed 61.0 (55.2, 67.3) Prevalence of B6 status categories‡   B6 deficiency (plasma PLP< 20 nmol/L)  3 (1.5) Suboptimal B6 status (plasma PLP= 20–30 nmol/L)  22 (10.9) Adequate B6 status (plasma PLP> 30 nmol/L)  177 (87.6) † Values presented as geometric mean (95% CI). ‡#Values presented as n (%). Abbreviations: B6, vitamin B6; PLP, pyridoxal 5’-phosphate  3.3.4! Secondary Objective: Predictors of Vitamin B6 Status 3.3.4.1! Bivariate Analysis Table 3-7 summarizes the differences in plasma PLP concentration by categories of demographic and lifestyle factors. Table 3-8 presents the differences in plasma PLP concentration by categories of demographic and lifestyle factors in non-users of supplemental B6, given that the use supplemental B6 showed a high association with B6 status in the overall sample. The unadjusted association of plasma PLP and continuous variables is shown in Table 3-9.  Plasma PLP concentration significantly differed between categorical groups of ethnicity, supplemental B6 use, and smoking status, and was significantly associated with BMI and dietary B6 intake. Women of South Asian ethnicity had significantly lower plasma PLP concentration (47.6 nmol/L) compared with women of European ethnicity (66.9 nmol/L). Users of supplemental B6 had significantly higher plasma PLP concentration compared to non-users of supplemental B6; mean plasma PLP concentrations were 111.5 and  64 48.5 nmol/L, respectively. Plasma PLP concentration was significantly different in non-users of supplemental B6 with different smoking status. Among non-users of supplemental B6, non-smokers had significantly higher plasma PLP concentration compared to former and current smokers. BMI was significantly associated with plasma PLP concentration; for every unit increase in BMI, plasma PLP decreased by 3.7%. In contrast, a 1mg increase in dietary B6 intake was significantly associated with a 32.3% increase in plasma PLP concentration.  Plasma PLP concentration was not significantly associated with education level, household income, physical activity level, OC use, age, or protein intake in bivariate analyses. Similarly, there was no significant difference in the prevalence of B6 deficiency or suboptimal B6 status or both combined compared to adequate B6 status based on any demographic or lifestyle factors. However, variables with borderline significance (P≤ 0.20) included education and total household income. Women of low education level had a higher prevalence of suboptimal B6 status (29%) compared to women of high education level (10%) (P= 0.16). Only in the supplemental B6 stratified bivariate analysis, the mean plasma PLP concentration was borderline lower in women of high income level (P= 0.19). The full model with plasma PLP concentration as the dependent variable was controlled for dietary B6 intake, ethnicity, BMI, education status, total household income status, smoking status, and supplemental B6 use. 65 Table 3-7Plasma Pyridoxal 5’- phosphate Concentration by Categories of Demographic and Lifestyle Factors in Young Adult Women in Metro Vancouver  Variables Participants, n Mean (95% CI), nmol/L P-value <20 nmol/L, n (%) P-value 20–30 nmol/L, n (%) P-value Overall 202 61.0 (55.2, 67.3)  3 (1.5)  22 (11)  Anemia        Hb ≥ 12g/dL* 166 62.0 (55.5, 69.2) 0.48 3 (1.8) 0.96 17 (10) 0.73 Hb < 12g/dL 36 56.4 (45.0, 70.8)  0  5 (14)  Ethnicity        European* 147 66.9 (59.2, 75.6) 0.002 2 (1.4) 1 14 (10) 0.44 South Asian 55 47.6 (41.2, 54.8)  1 (1.8)  8 (15)  Education        Low 7 57.7 (28.5, 116.9) 0.84 0 1 2 (29) 0.16 High* 195 61.1 (55.3, 67.5)  3 (1.5)  20 (10)  Household income        Low 80 59.5 (52.1, 68.0) 0.55 1 (1.3) 1 7 (9) 0.55 High* 103 63.6 (55.3, 73.0)  2 (1.9)  15 (13)  Physical activity level        Low 8 44.4 (37.8, 52.3) 0.42 0 1 0 0.70 Medium 110 60.6 (53.0, 69.4)  2 (1.8)  13 (12)  High 82 63.8 (54.5, 74.8)  1 (1.2)  9 (11)  Oral contraceptive use        No* 144 59.9 (53.5, 67.1) 0.59 2 (1.4) 1 17 (12) 0.68 Yes 58 63.6 (52.0, 77.8)  1 (1.7)  5 (9)  Supplemental vitamin B6 use        No* 146 48.5 (45.0, 52.3) <0.001 3 (2.1) 0.56 18 (12) 0.42 Yes 56 111.5 (87.2, 139.9)  0  4 (7)  Current or Former Smoker        No* 174 61.1 (55.0, 67.8) 0.92 2 (1.2) 0.36 18 (10) 0.75 Yes 28 60.2 (44.0, 82.3)  1 (3.6)  4 (14)  *Referent category. Difference in plasma pyridoxal 5’-phosphate concentration between groups was assessed for dichotomous variables by two sample t-test and for categorical variables by one-way ANOVA. Difference in prevalence of plasma pyridoxal 5’-phosphate concentration < 20 nmol/L and 20–30 nmol/L was assessed by chi-square test. Abbreviation: Hb, hemoglobin.  66 Table 3-8 Plasma Pyridoxal 5’-phosphate Concentration by Categories of Demographic and Lifestyle Factors in Non-users of Supplemental Vitamin B6 in Young Adult Women in Metro Vancouver  Variables Participants, n Mean (95% CI), nmol/L P-value <20 nmol/L, n (%) P-value 20–30 nmol/L, n (%) P-value Overall 146 48.5 (45.0, 52.3)  3 (2.1)  18 (12)  Anemia        Hb ≥ 12g/dL* 119 48.0 (44.4, 52.0) 0.60 3 (2.5) 0.93 14 (12) 0.91 Hb < 12g/dL 27 50.7 (39.9, 64.4)  0  4 (15)  Ethnicity        European* 104 51.5 (46.8, 56.7) 0.014 2 (1.9) 1 12 (12) 0.86 South Asian 42 41.8 (37.2, 46.9)  1 (2.4)  6 (14)  Education        Low 5 51.8 (19.9, 134.8) 0.75 0 1 2 (40) 0.22 High* 141 48.4 (44.9, 52.1)  3 (2.1)  16 (11)  Total household income        Low 60 51.3 (45.6, 57.8) 0.19 1 (1.7) 1 5 (8) 0.32 High* 71 46.2 (42.1, 50.6)  2 (2.8)  11 (16)  Physical activity level        Low 7 44.9 (37.2, 54.2) 0.82 0 0.88 0 0.44 Medium 80 48.1 (42.9, 53.9)  2 (2.5)  12 (15)  High 57 49.8 (44.7, 55.5)  1 (1.8)  6 (11)  Oral contraceptive use        No* 107 48.9 (44.7, 53.4) 0.76 2 (1.9) 1 14 (13) 0.86 Yes 39 47.6 (40.5, 55.9)  1 (2.6)  4 (10)  Current of former smoker        No* 127 50.0 (46.0, 54.3) 0.047 2 (1.6) 0.85 14 (11.0) 0.39 Yes 19 39.8 (33.1, 47.9)  1 (5.3)  4 (21)  *Referent category. Difference in plasma pyridoxal 5’-phosphate concentration between groups was assessed for dichotomous variables by two sample t-test and for categorical variables by one-way ANOVA. Difference in prevalence of plasma pyridoxal 5’-phosphate concentration < 20 nmol/L and 20–30 nmol/L was assessed by chi-square test.  Abbreviation: Hb, hemoglobin.  67 Table 3-9 Unadjusted Association of Continuous Variables with Plasma Pyridoxal 5’-phosphate in Young Adult Women in Metro Vancouver Variables Participants, n Unadjusted % change in plasma PLP (95% CI)* P-value Age, y 202 -0.24 (-2.6, 2.2) 0.85 BMI, kg/m²  202 –3.7 (–6.3, –1.0) 0.007 Protein intake, g/day 184 0.25 (-0.25, 0.76) 0.32 Dietary B6 intake, mg/day 184 32.3 (4.8, 67.0) 0.019 * The percentage change in plasma PLP concentration was calculated by ( !"# − 1) *100% determined by simple linear regression. Abbreviations: B6, vitamin B6; BMI, body mass index; PLP, pyridoxal 5’-phosphate  3.3.4.2! Multivariable Regression Model Relative dietary B6 intake, BMI, ethnicity and the use of supplemental B6 were significant predictors of plasma PLP concentration (Table 3-10). Relative dietary B6 intake and the use of supplemental B6 were positively associated with plasma PLP concentration; BMI and South Asian ethnicity were inversely associated with plasma PLP concentration. The model explained 32% (R²) of the variance in plasma PLP concentration.  Compared to a dietary B6 intake of <1.1 mg/day, dietary B6 intake between 1.1−1.7 mg/day did not significantly associate with a change in plasma PLP concentration. However, having a dietary B6 intake >1.7 mg/day compared to dietary B6 intake <1.1 mg/day was associated with a 29.3% increase in plasma PLP concentration adjusting for BMI, ethnicity and supplemental B6 use (p= 0.048). In the adjusted model, every unit increase in BMI was estimated to decrease plasma PLP concentration by 2.7%; women taking supplemental B6 are expected to have 114.5% higher plasma PLP; and South Asian ethnicity was associated with 21% lower plasma PLP concentration compared to women of European ethnicity.  68  Table 3-10 Predictors of Plasma Pyridoxal 5’-phosphate Concentration in Young Adult Women in Metro Vancouver Variables Unadjusted % change in plasma PLP (95% CI)* Unadjusted P-value Adjusted % change in plasma PLP (95% CI)* Adjusted P-value Relative B6 intake     Q2 (1.1–1.4 mg/day) 7.7 (–20.0, 45.0) 0.62 9.0 (–15.8, 41.2) 0.51 Q3 (1.4–1.7 mg/day) 27.5 (–5.3, 71.6) 0.11 23.1 (–4.4, 58.6) 0.11 Q4 (>1.7 mg/day) 35.6 (0.7, 82.4) 0.045 29.3 (0.3, 66.7) 0.048 BMI, kg/m² –3.7 (–6.3, –1.0) 0.007 –2.7 (–5.1, –0.2) 0.034 South Asian ethnicity† –28.9 (–33.6, –24.0) 0.002 –21.1 (–35.7, –3.1) 0.024 Supplemental B6 use 127.7 (115.6, 140.8) <0.001 114.5 (75.6, 162.0) <0.001 Number of observation = 184, Model P <0.001, R²: 0.32, Adjusted R²: 0.29.  * The percentage change in plasma PLP concentration was calculated by (!"# − 1)*100%. † Referent category: European ethnicity Abbreviations: B6, vitamin B6; BMI, body mass index; PLP, pyridoxal 5’-phosphate  3.4! Discussion Given the data on biochemical B6 status of Canadian women are very limited and the relationship between demographic, dietary, and lifestyle factors and plasma PLP had not been assessed to date, I conducted a secondary analysis in a convenience sample of 202 healthy young Canadian adult women. The sample revealed a combined prevalence of B6 deficiency and suboptimal B6 status of 12.4%. BMI, South Asian ethnicity, relative dietary B6 intake, and the use of supplemental B6 were found to be the significant predictors of plasma PLP concentration. 3.4.1! Prevalence of Vitamin B6 Deficiency and Suboptimal Vitamin B6 Status The prevalence of B6 deficiency (1.5%) in this sample of 202 young adult women was much lower than the 40% prevalence of B6 deficiency reported for women aged 21−44  69 years in the NHANES 2003−2004 [22]. This large discrepancy may partly be due to the high socioeconomic status of the women in this study compared with participants of the NHANES, a representative population-based survey in the US. Low socioeconomic status has been associated with low B6 status [24,25] and overall poorer nutritional intake [138]. Education and income status accounted for 6% of the variation in plasma PLP in the NHANES 2003-2006 [24]. The discrepancy between this study and the NHANES may also be attributed to different analytical methods employed for quantification of plasma PLP. The enzymatic assay used for the NHANES 2003-2004 samples has been reported to underestimate plasma PLP concentrations to a degree that would result in a doubling of the prevalence of B6 deficiency compared to the HPLC method we employed [139].  Compared with one of the few available Canadian studies on biochemical B6 status, the recent Alberta Pregnancy Outcomes and Nutrition cohort study found no B6 deficiency among the 119 first-trimester pregnant women, and that only 1 out of the 528 second-trimester pregnant women was B6 deficient [21]. The prevalence of suboptimal B6 status was not reported [21]. Like this study, the Alberta Pregnancy Outcomes and Nutrition cohort is comprised of women of high socioeconomic status and the lack of B6 deficiency observed is likely due to the high rate (> 94%) of multivitamin supplement use throughout pregnancy [21]. In the CCHS in 2004, 18% of Canadian adult women did not meet the EAR of B6 from dietary sources [20]. However, the prevalence of B6 deficiency was much lower than the  70 prevalence that I expected based on the CCHS in 2004. The discrepancy may be explained by the fact that the CCHS in 2004 reported the B6 intake solely from dietary sources without incorporating the intakes from supplements. The prevalence of inadequate dietary B6 intake from the CCHS in 2004 might not adequately reflect the prevalence in Canadian women.  Vitamin B6 status has been inversely associated with the risk of several chronic diseases, including cardiovascular disease [4–9], colorectal cancer [10,11,106], breast cancer [12,13], lung cancer [14,107] and ovarian cancer [140]. In this study, 11% of our participants had suboptimal B6 status. In the study of Ye et al., a cross-sectional study in Boston reported 18% of suboptimal B6 status among Puerto Rican women aged 45−75 years (n= 1236) [25]. The participants in the study of Ye et al. were recruited for a longitudinal study focusing on relationships between stress, nutrition, and chronic health conditions. They had an overall lower education level (48% had education lower than grade 9th) and lower income (29% lived in poverty) in that study [25]. The lower socioeconomic status of participants in the study of Ye et al. may explain the higher prevalence of suboptimal B6 status compared with this study.  In light of the limited data on B6 status in Canadians [20,21] and the findings of this study which observed a substantial rate of suboptimal status in a cohort of healthy women of high socioeconomic status, a representative, population-based study to determine the B6 status of Canadians is timely and crucial. It is possible that a higher prevalence of B6  71 deficiency and suboptimal B6 status would be observed in a representative sample of Canadian women compared to this study. 3.4.2! Predictors of Plasma Pyridoxal 5’-phosphate 3.4.2.1! Use of Supplemental Vitamin B6 The use of supplemental B6 was a strong predictor of plasma PLP concentration both before and after multivariable adjustment. This is consistent with large-scale studies that reported higher plasma PLP concentrations and a lower prevalence of B6 deficiency in nutritional supplement users [22] and supplemental B6 users [23] compared with non-users. In this sample, 48% of women used nutritional supplements (i.e., any type of vitamin or mineral supplement) of whom 58% were retrospectively categorized as supplemental B6 users. The prevalence of nutritional supplement use in this sample (48%) was higher than the prevalence reported in the CCHS in 2004 [141] with 35% of Canadian women aged 19−30 years reporting nutritional supplement use. Although data on the type of nutritional supplement were not collected in the CCHS, it can be hypothesized that the prevalence of supplemental B6 use in Canadian young adult women is less than 30%. Given a potentially lower prevalence of supplemental B6 use in Canadian women and considering that B6 intake is a major contributor of plasma PLP concentration [22,23], I hypothesize a higher prevalence of B6 deficiency and suboptimal B6 status in a representative sample of Canadian women compared to the combined prevalence of 12.4% found in this study.  72 3.4.2.2! Dietary Vitamin B6 Intake Relative dietary B6 intake was not a strong predictor of plasma PLP concentration both before and after multivariable adjustment. The weak association may partially be due to the use of a semi-quantitative FFQ. The validation study of the FFQ reported an 11% underestimation of relative dietary B6 intake compared to the use of dietary recalls [135]. Although the FFQ was validated to assess micronutrient intakes in the Canadian population [135], it is possible that it was unable to adequately reflect South Asian diet. Further, under-reporting appears to be a factor, as a 1566 kcal/day average daily energy intake is lower than would be anticipated. In the NHANES 2003−2004, a stronger association between total B6 intake (from food sources and supplements combined) and plasma PLP (r = 0.32, P < 0.001) was observed [22]. We are unable to report quantitative intake of B6 from supplements due to limitations of the secondary analysis. The weaker association between plasma PLP and dietary B6 intake in this study compared to the NHANES data might be explained by our data reflecting dietary B6 from food alone and not supplemental B6. 3.4.2.3! Ethnicity This study is the first to report that women of South Asian ethnicity may have lower plasma PLP concentrations compared with women of European ethnicity. The inverse association between South Asian ethnicity and plasma PLP concentrations remained significant after adjustment for relative dietary B6 intake, BMI, and supplemental B6 use.  73 The difference in B6 status between South Asian and European women was not related to the consumption of different food sources of B6; meat and meat alternatives were the main dietary B6 sources in these healthy adult women and there was no significant difference in the consumption of meat and meat alternatives between European and South Asian. However, this finding should be interpreted with caution since the FFQ was not designed for specific ethnicity and might miss to capture some foods in the South Asian diet. The South Asian population was only 56 participants, compared to the European population of 146 in this study; nonetheless, the sample size was sufficient to give over 80% of power with a small effect size of 0.2 and a significance level of 0.05 in the multiple linear regression model. Ethnicity has also been shown to correlate with nutritional biomarker levels of other micronutrients (e.g. ferritin, folate) in NHANES 1999−2002 [142]. Therefore, I recommend future studies to assess ethnicity when investigating B6 status. 3.4.2.4! Body Mass Index Body mass index was inversely associated with plasma PLP concentration after adjustment for relative dietary B6 intake, supplemental B6 use, and South Asian ethnicity. An inverse relationship between BMI and plasma PLP was also reported in the NHANES; every 25% increase in BMI was associated with a 13% decrease in plasma PLP [24]. The volumetric dilution of the blood was suggested as a potential underlying mechanism for the linkage between increasing body mass and decreasing concentrations of B6 as well as other  74 B-vitamins [89]. In addition, plasma PLP has been inversely associated with circulating concentration of CRP, a marker of systemic inflammation that is elevated in obesity [76,90]. A majority of the evidence proposed that inflammation causes increased utilization of B6 in sites of inflammation, which leads to the redistribution of B6 and low circulating PLP concentrations which can possibly induce B6 deficiency in the human body [112,113,116]. 3.4.2.5! Other Variables Compared to other studies, we did not find OC use to be a significant predictor of B6 status. In recent literature, lower-dose OC use was associated with low B6 status [66]. In the NHANES 2003–2004, plasma PLP concentration was significantly lower in OC users compared with non-users [22]. Also in their study, the prevalence of plasma PLP concentration < 20 nmol/L were 40% in former OC users and 78% of current users [22]. It was suggested that this may be due to the disruption of tryptophan metabolism by estrogen, independent of B6 status [67]. In this study, the effect of OC use on plasma PLP concentration was small and we had an insufficient sample size to detect a statistically significant difference. This sample also lacked power to detect associations with other variables including physical activity (only 8 participants in low physical activity level) and smoking (only 19 participants were former or current smokers).  75 3.4.3! Strengths and Limitations This study is the first study to report on the biochemical B6 status in non-pregnant young adult women in Canada. In addition, we are the first study to report an association of low plasma PLP with South Asian ethnicity, after having controlled for other predictors of B6 status, including relative dietary B6 intake, supplemental B6 use, and BMI.  While there are important strengths to this study, there are a number of limitations; first, the recruitment of a convenience sample of relatively healthy women of high socioeconomic status. Over 71% of our study participants obtained a bachelor’s degree or higher. A comparatively lower proportion of 35% of women aged 25−64 years in Metro Vancouver reported this high level of education based on results from the National Household Survey [143]. Yet, studies have found that B6 status differed between income levels but not education levels [24,25]. The higher socioeconomic status and the inclusion of relatively healthy participants in this study compared to the overall population of Canadian women are factors that limit the generalizability of the findings of this study.  Second, because this study was a secondary analysis, I could only retrospectively obtain the information on the use of supplemental B6 that did not allow quantitative assessment of supplemental B6 intake. This might be the one of the reasons why a weak correlation between dietary B6 intake (from food alone) and plasma PLP was observed if the participants actually consumed high amount of B6 from supplements. Additionally, the use of  76 a semi-quantitative FFQ limits the explanations on dietary B6 intake since it only gives an estimation of usual dietary intakes and is a less preferable tool to assess dietary intakes compared with 24-hour recall. The mean dietary B6 intake from this study was incomparable with the dietary B6 intakes reported in the CCHS in 2004 given that they used 24-hr recalls to calculate the mean dietary B6 intake. The findings on dietary B6 intake in this study may not provide a complete perspective on its impact on B6 status or plasma PLP concentration using FFQ without including quantitative supplemental B6 intake.  Third, the four variables in the multiple linear regression only explained 32% of the variability in plasma PLP. The results might be confounded by unexplained biological factors or genetic modifiers, specifically genetic variants might have influenced the findings, which I did not control for. One recent study reported some variants in tissue-nonspecific alkaline phosphatase gene to be significantly associated with plasma PLP concentration, but the clinical implications of these variants were unclear as the study did not investigate whether there was a functional change in B6 status [144].  Another limitation of this study was the use of plasma PLP as the single biomarker to assess B6 status. I used plasma PLP because it is currently the only established biomarker with cut-offs to define B6 deficiency [2]. However, plasma PLP only reflects the circulating concentration of B6 and is influenced by several factors, such as inflammation, serum albumin, and alkaline phosphatase concentrations [38]. Some substrates in B6-dependent  77 pathways can serve as functional indicators that reflect intracellular B6 status, for example, plasma cystathionine [48,51,52] and plasma kynurenines [38,41,42]. The derivation of reference intervals for sensitive functional biomarkers is crucial for evaluation of intracellular B6 status.  3.5! Conclusion A 12.4% combined prevalence of B6 deficiency and suboptimal B6 status was found in this convenience sample of 202 healthy young adult women in Metro Vancouver. Preconceptional B6 adequacy is crucial for healthy pregnancy outcomes; and women with higher BMI and South Asian ethnicity might be more likely to have low B6 status. The lower prevalence of B6 deficiency and suboptimal B6 status in these young women compared to the reports from representative samples in other countries may be due to the high socioeconomic status in this study. Given the central roles of B6 in key biological functions and health, more research is needed to determine B6 status in the general Canadian population.  78 Chapter 4:!Vitamin B6 Status in Older Adult Women in Metro Vancouver 4.1! Introduction The research described in this chapter aimed to understand the prevalence of vitamin B6 (B6) deficiency and predictors of B6 status using plasma pyridoxal 5’-phosphate (PLP) as the primary status indicator in a population different from the young adult women discussed in Chapter 3. A cross-sectional study recruiting older adult women in Metro Vancouver with convenience sampling methods was designed to achieve the following objectives. The primary objective was to determine the biochemical B6 status and the prevalence of B6 deficiency and suboptimal B6 status in older adult women aged 51−70 years in Metro Vancouver. High prevalence of inadequate dietary B6 intake was reported among older adult women in the Canadian Community Health Survey (CCHS) 2004 [20]; 19.4% of older adult women aged 51−70 years did not meet the recommendations for dietary B6 intake, compared to 9.6% of young adult women aged 19−35 years. Thus, I hypothesized a higher prevalence of low B6 status in older adult women compared with the findings in my previous study in young adult women. This study aims to provide data on the prevalence of B6 deficiency in a sample of Canadian older adult women.  The secondary objective was to identify demographic, dietary, and lifestyle predictors associated with B6 status in a convenience sample of Vancouver older adult women. In my previous study in young adult women, I identified South Asian ethnicity and higher BMI to  79 be associated with lower plasma PLP. This study aims to explore other variables that are unique in older adult women, such as menopausal status and hormone replacement therapy. The results will provide more data to help with the identification of potentially vulnerable population groups for low B6 status and related health consequences in Canada.  4.2! Methods 4.2.1! Study Design  For this descriptive cross-sectional study, the target sample size was 200 healthy, older adult women aged 51−70 years. The age group of 51−70 years was selected because the CCHS in 2004 reported 19.4% of dietary B6 intake below the EAR in women aged 51−70 years [20]. The high prevalence of inadequate dietary B6 intake among Canadian older adult women indicates that this population group may be at risk of being B6 deficient. Enrolment occurred between October 2015 and October 2016 in Metro Vancouver. Participation in the study was voluntary. Women who were interested in the study contacted the research team and were scheduled for a clinic visit after their eligibility was checked. Participants were required to attend one single study visit (~ 1.5 hours) to provide their written consents, anthropometric information, a fasting blood sample, and demographic and dietary intake data. The Clinical Research Ethics Board at UBC (#H15-01937) and the Vancouver Coastal Health Ethics Board (#V15-01937) gave ethical approval for conduct of this study.  80 4.2.2! Study Participants 4.2.2.1! Sample Size With an expected 15% prevalence of low B6 status at a 95% confidence level, we needed a sample size of 196 to achieve a 5% margin of error for the study of older adult women. The expected 15% prevalence of low B6 status was chosen based on the result of my previous study in young adult women in Metro Vancouver in which a 12.4% prevalence of low B6 status was identified. The equation used to calculate the sample size is shown in Equation 1. Equation 1. Sample Size Calculation n = (()*)*×,×(#-,).*  with E: margin of error; p: estimated planned proportion; /)*: 100(1 − α2) percentile of the standard normal distribution 4.2.2.2! Inclusion and Exclusion criteria  The study population consisted of older adult women aged 51−70 years residing in Metro Vancouver. Eligibility of potential participants was assessed using a screening questionnaire (Appendix A), administered over the phone or through email. To participate in the study, women were required to meet the following inclusion criteria: I.! Aged between 51 and 70 years II.! Self-identified healthy  81 III.! Reside in Metro Vancouver Women were excluded based on the following criteria: I.! Major chronic health condition for which they are receiving treatment or medication, for example hydralazine, phenelzine, tranylcypromine (full list of medications can be found in section 5 of Appendix A). II.! Using medications that interact with B6 status, or recreational drugs III.! Suffer from an active gastrointestinal condition or have undergone gastric bypass 4.2.3! Recruitment Participants were recruited by passive and active methods in Metro Vancouver. Passive recruitment methods include: distributing study flyers at various places, posting an online advertisement on websites and social media, sending broadcast emails through different list-serves, and placing an advertisement in newspapers. Paper flyers in English and Chinese (Appendix B) and brochures (Appendix C) were placed in areas of high public traffic (e.g., coffee shops, libraries, community centers, bus stops, grocery stores, pharmacies, senior centers, doctor’s offices, BC Women’s Hospital, Women’s Clinic at Vancouver General Hospital, UBC Hospital, UBC campus buildings). Online advertisements were placed through Vancouver Costal Health Research Institute website under active clinical trials, Western Nutrition Research Centre website under current recruiting studies, UBC Psychology Graduate Student Council website under paid participants studies list, craigslist, and  82 Facebook. Broadcast emails were sent out through academic and professional email listservs (i.e., BC Children’s Hospital Research Institute listserv, Vancouver Coastal Health Research Institute listserv, UBC Land and Food System Staff and Alumni listserv, UBC Food Nutrition and Health listserv, CRCNet listserv, UBC Community Centre listserv). Newspaper advertisements were placed in Metro News, 24hr Vancouver, Vancouver Courier, Westender, Richmond News, Burnaby Now, and New West Record. In addition, an article was published about the study in the Vancouver Coastal Health Research Institute e-newsletter. Participants were also actively recruited from recruitment booths at community events (e.g., Strawberry Festival 2016), farmers’ markets (e.g., UBC Farmers’ Market), shopping malls (e.g., Richmond Centre), and at a local conference (Dietitian Canada BC Spring Conference 2016). I also gave a presentation about the study and its significance at the Tapestry Masterclass Speaker Series and at a community nutritionists meeting. 4.2.4! Study Visit Procedure Prior to the study visit, an information package including appointment time and location, a primary informed consent form (Appendix D), an optional consent form for tissue banking (Appendix E), and a study visit protocol (Appendix F) was sent to the participants through email or postal mail at least 24 hours prior to their study visit. Participants attended one clinic visit in Vancouver, BC, at either the UBC Western Nutrition Research Center in the Food, Nutrition, and Health building or at the BC Children’s Hospital Clinical Research and  83 Evaluation Unit. Before the participants gave their written consents, I ensured that they had read and understood the informed consent and that there were no outstanding questions about participant responsibilities. Each participant was assigned an identification number beginning with 001 that was recorded on all questionnaires and biological samples.  After obtaining written consents from the participants, I or a trained research team member measured the height, weight, waist, and hip circumferences, and blood pressure of the participant, with light clothing and no shoes using a stadiometer, digital scale, measuring tape, and blood pressure monitor, respectively. All measurements were performed and recorded in duplicate. Participants were asked to bring in nutritional supplement bottles or clear pictures of the bottles to provide information on their supplement use in the past month. I recorded the information from all the participants including descriptive data (i.e. brand name, product name, dose, and frequency) and quantitative values for B6, folate and vitamin B12 supplement intake (i.e., mg or µg per tablet). Following the anthropometric measurements and supplement use recording, 24 mL fasting (~10 hours) venous blood samples was drawn from participants by a certified research phlebotomist. After the blood draw, participants were brought to a quiet room to complete a demographic questionnaire (Appendix G) and a diet history questionnaire (DHQ) adapted for Canadian populations from the National Institutes of Health (C-DHQ II) [145]. Each participant received a basic, standard instruction for the filling of the questionnaires by  84 research staff. Research staff periodically checked that there were no questions regarding the questionnaires.  Once the participants finished the questionnaires, research staff manually checked each questionnaire to ensure that there was no missing answer or errors. The participants were provided a $20 gift card to a grocery store to defray the costs of transportation and received free breakfast. After all analyses had been completed, results of B6, folate, and vitamin B12 intake and blood measurement were sent to the participants in the form of an ethics approved report, presenting the individual’s results in reference to the Dietary Reference Intakes [2] and vitamin status based on cut-offs of adequacy (Appendix H).  A flowchart demonstrating the study visit procedure is shown in Figure 4-1.   Written!informed!consent!was!obtained.Anthropometric!measurements!and!information!on!supplement!use!were!collected.24mL!fasting!blood!sample!was!collected.!Participants!answered!the!demographic!questionnaire!and!diet!history!questionnaire.Questionnaires!were!manually!checked!by!research!staff.!Figure 4-1 Procedure for the Study Visits  85 4.2.5! Demographic and Food Frequency Questionnaires The demographic questionnaire consisted of three modules: anthropometric measurements, socioeconomic and lifestyle and supplement use. Variables related to socioeconomic status and lifestyle factors included the collection of data about age, ethnicity, location, education, income, smoking status, lactose intolerance, menstrual or menopausal status, and hormone and contraceptive use. For ethnicity, participants were asked to identify themselves as either European, South Asian, Chinese or other ethnicities. South Asian and Chinese ethnicity were specified because they are the top two largest minority groups in Canada [81]. Menstrual or menopausal status was self-reported by the participants. Smoking status was categorized as non-smoker, former smoker, current occasional smoker (1–9 cigarettes/day), current regular smoker (10–19 cigarettes/day) or current frequent smoker (≥20 cigarettes/day) and further grouped as non-smoker, former smoker or current smoker. The last part of the demographic questionnaire, history of nutritional supplement use in the past month, was recorded by me. I asked questions such as ‘how often do you use this supplement’ and ‘how many tablets do you take each time’ to obtain information including the brand name, product name, dosage, and frequency of each supplement and quantitative values for B6, folate and vitamin B12 supplement intake (i.e. mg or µg per tablet).  Data from the demographic questionnaires was manually and doubly entered into an Excel™ spreadsheet (Microsoft Corporation, 2013) and then checked for disagreement between  86 entries. Repeated anthropometric measurements for each participant were averaged. Few variables were created with further calculations and are explained in Table 4-1.  Table 4-1 Calculations for Selected Variables in Vitamin B6 Status of Older Adult Women in Metro Vancouver Variable Calculation BMI Weight6(kg)/6height(m)^266 Waist and hip ratio Waist circumference (cm) / hip circumference (cm) Total household income Lowest  [132]  less than $15,000 if 1 or 2 people less than $20,000 if 3 or 4 people less than $30,000 if 5 or more people  Lower-middle  $15,000 to $29,999 if 1or 2 people $20,000 to $39,999 if 3 or 4 people $30,000 to $50,000 if 5 or more people  Upper-middle  $30,000 to $59,999 if 1 or 2 people $40,000 to $79,000 if 3 or 4 people $60,000 to $79,999 if 5 or more people  Highest  $60,000 or more if 1 or 2 people $80,000 if 3 or more people Vitamin B6 intake from supplement Dose of supplement (mg) * number of doses per day * daily frequency of supplement use (i.e. 1 times per day)   The C-DHQ II is a food frequency questionnaire which contains 165 food-item questions that were adapted from the US DHQ II for Canadian populations [145,146]. The food list and the database used for calculating the nutrient intakes were based on analyses of 24-hour recall reported in the CCHS data in 2004 [146]. The C-DHQ II we used was the paper-based, ‘Past year, with portion size’ version which estimates the usual frequency and portion size of foods consumed in the past 12 months [145]. C-DHQ II has not yet been validated. The older version of the US DHQ I has been validated to measure nutrient intakes in adults with 24-hour recalls [147,148] although some underreporting on total energy intakes  87 and protein intakes were also identified [149]. Given the similarities between the US DHQ I and C-DHQ II, the C-DHQ II is thought to have moderate ability to capture diet with a significant underestimation of energy and protein intake [146]. The completed C-DHQ IIs were scanned and sent to Alberta Health Services, CancerControl Alberta, the Department of Cancer Epidemiology and Prevention Research for the processing of dietary information. Intakes of 34 nutrients were analyzed using Diet*Calc Analysis Program (Version 1.5.0, National Cancer Institute, 2014). Participants with complete blank page(s) or energy intakes assumed to be biologically implausible (<600 kcal/day or 3500 kcal/day) [147,150] were excluded from the analysis. 4.2.6! Laboratory Methods 4.2.6.1! Initial Blood Sample Processing  Research phlebotomists collected 24 mL of blood from each participant in one 4 mL serum and two 10 mL lithium heparin vacutainers using butterfly needles. The serum tube was left to sit upright for at least 30 minutes at room temperature. The lithium heparin tubes were immediately placed on ice and covered to prevent light exposure. I performed most of the initial sample processing and the procedure is explained below.  For each participant, three 100 µL aliquots of whole blood were pipetted from the lithium heparin tube into 1 mL of freshly prepared 10% (w/v) L-ascorbic acid solution in labelled amber micro-tubes. The amber micro-tubes were vortexed and incubated in either a  88 heating water bath or heating block at 37°C for three hours. This allowed the conversion of red blood cell folate polyglutamates to monoglutamates. Following the incubation, the samples were stored at –80°C. For the analyses of hemoglobin concentration and hematocrit level, 200µL of whole blood were pipetted from the lithium heparin tubes into a labelled micro-tubes. From the micro-tube, 13 µL of whole blood was pipetted to a piece of parafilm and automatically draw a precise amount of blood using a micro-cuvette. The micro-cuvette was then placed in a HemoCue® Hb 301 machine (HemoCue®, Sweden) for the analysis of hemoglobin concentration. The hemoglobin concentrations were immediately read and recorded in a research lab notebook. Two micro-hematocrit capillary tubes were filled with whole blood from the micro-tube to about 3/4 full, sealed with clay, and centrifuged for 10 minutes. Hematocrit levels were read using the micro-hematocrit tube reader and recorded in the research lab notebook. Hematocrit measurements were averaged. The lithium heparin tubes were then centrifuged at 3000 rpm for 15 minutes at 4°C. Five 500 µL and five 1 mL plasma aliquots and two buffy coat aliquots were pipetted into labelled micro-tubes and stored at –80°C. The tubes containing the red blood cells were filled with room temperature, pH 7.4 phosphate buffered saline (PBS), gently inverted, and centrifuged for 15 minutes at 3000 rpm at 4°C. The supernatant and leftover buffy coat were discarded and the tubes were refilled with PBS and spun again for 10 minutes at 2000 rpm at 4°C. This process was repeated two times. After the final spin and the supernatant and  89 leftover buffy coat were discarded, five 500 µL and three 1 mL washed red cells aliquots were pipetted into labelled micro-tubes and stored at –80°C. The serum tubes were centrifuged at 3000 rpm for 15 minutes at 4°C after sitting at room temperature for at least 30 minutes and maximum 90 minutes. Three 500 µL serum aliquots were pipetted into labelled micro-tubes and stored at –80°C. A flowchart demonstrating the procedure is shown in Figure 4-2.    90 Figure 4-2 Procedure for Initial Blood Sample Processing  91 4.2.6.2! Biochemical Vitamin B6 Status Assessment  Blood samples of 222 participants were available for the analysis of plasma PLP. Plasma PLP was quantified using isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) by a modified method according to Midttun et al. [151]. The advantages of LC-MS/MS over HPLC are high precision allowing singlet analysis and higher sensitivity leading to smaller blood volume requirements. The sample extraction consisted of a protein precipitation step with trichloroacetic acid after the addition of the internal standard. Sample extracts were injected into an LC system (Agilent 1260, Agilent Technologies). Compounds were separated by a C18 column (2.1 x 50 mm, 3.5 µm). The mobile phase consisted of (A) 10mM ammonium and (B) 10mM ammonium in 95% acetonitrile using a gradient run. The affluent was directed into an MS/MS system (API4000, SCIEX Pte). Plasma PLP was quantified with an 8-point calibration curve (2.5–320 nmol/L) made using PLP (Sigma Aldrich) as calibrator and deuterium-labeled PLP (Cambridge Isotope Laboratories) as internal standard. Three external quality control samples manufactured by ClinChek 8873 (RECIPE, IRIS Technologies International) were quantified with every analysis. Mean ± SD serum PLP concentrations of the three controls (CCI – CCIII) were within their acceptable ranges (mean; range): CCI 58.5 ± 2.2 (61.9; 49.4-74.5) nmol/L, CCII 95.0 ± 1.1 (97.1; 77.7-117) nmol/L, and CCIII 139.5 ± 0.7 (138; 110-165) nmol/L; the inter- 92 assay CVs for the quality control samples over the three analytical days were 4%, 1% and 1%, respectively. The cut-off values used for B6 deficiency in this thesis are as follows: plasma PLP  > 30 nmol/L for adequate B6 status, plasma PLP = 20–30 nmol/L for suboptimal B6 status [3], and plasma PLP < 20 nmol/L for B6 deficiency [2,3]. 4.2.7! Final Variables Included in Analysis Final variables from all questionnaires and biological assessment included in the analysis are summarized in Table 4-2.  93 Table 4-2 Variables and Coding Method Included in Vitamin B6 Status of Older Adult Women in Metro Vancouver Variable Type Unit or coding Height Continuous cm Weight Continuous kg Age Continuous years Waist circumference Continuous cm Hip circumference Continuous cm Waist and hip ratio Continuous N/A Body mass index Continuous kg/!" Blood pressure Continuous mm Hg Ethnicity Categorical 1=European, 2=Chinese, 3=South Asian, 4=Other Menopause Categorical 0=No, 1=Yes Hormone use Categorical 0=No, 1=Former, 2=Current Oral Contraceptive use Categorical 0=No, 1=Former, 2=Current Nutritional supplement  Categorical 0=No, 1=Yes Supplemental B6 use Categorical 0=No, 1=Yes Smoker Categorical I. 0=Never, 1=Former, 2=Current  Categorical II. 0=Non-smoker, 1=Former smoker, 2=Current occasional smoker (1–9 cigarettes/day), 3=Current regular smoker (10–19 cigarettes/day), 4=current frequent smoker (≥20 cigarettes/day) Income Categorical I. 1=Low, 2=Low-middle 3=Middle-high, 4=High  Categorical II. 0=High, 1=Low Education Categorical I.  1=Less than secondary school education, 2=Secondary school diploma, 3=Post-secondary education, 4=Bachelor’s degree, 5=University degree or >than bachelor’s degree  Categorical II. 0=High, 1=Low Anemia Categorical Hemoglobin ≥12=No, hemoglobin <12=Yes Plasma PLP Continuous nmol/L  Categorical > 30=adequate status, 20-30=suboptimal status, < 20=deficiency Energy intake Continuous kcal Protein intake Continuous g Dietary B6 intake Continuous mg Supplemental B6 intake Continuous mg Abbreviations: B6, vitamin B6; PLP, pyridoxal 5’-phosphate  94 4.2.8! Statistical Analyses All statistical analyses were performed using R software (3.1.2 windows version).  Variables were described depending on the level of measurement. Continuous variables were examined for normality by visual histogram assessment. Normally distributed variables were reported as the arithmetic mean ± SD. Non-normally distributed variables were log-transformed and reported as geometric mean (95% CI). Categorical variables were presented by a frequency table. Statistical significance was set at a two-sided P-value of < 0.05. The primary objective was to determine the biochemical B6 status and the prevalence of B6 deficiency and suboptimal B6 status in older adult women in Metro Vancouver. The primary outcome of this study, B6 status, was described using plasma PLP and the cut-offs for adequate B6 status, suboptimal B6 status, and B6 deficiency, as described above. Plasma PLP concentration was log-transformed to carry out the statistical analyses and was presented as geometric mean (95% CI). The prevalence of B6 deficiency and suboptimal status was presented as frequency (%).  The secondary objective was to identify demographic, dietary, and lifestyle predictors associated with B6 status, using plasma PLP as the status indicator. To identify the predictors of B6 status, a multiple linear regression model was created with plasma PLP concentration as the dependent variable. For example:  Multiple linear regression model e.g. Plasma(PLP(concentration = (23 (+(25 66(89:;<= +(2" 6>? +(2@ AB= +⋯ The method for multivariable model construction is presented below: Bivariate analysis was conducted to identify variables associated with plasma PLP. If the P-value from the bivariate analysis was ≤0.2, the variable was included in the full model. A two-sample t-test was used for dichotomous variables, including education level, total household  95 income level, OC use, hormone therapy use, supplemental B6 use, and smoking. One-way ANOVA followed by Tukey’s Honest Significance test was used for categorical variables with more than two levels including ethnicity. Simple linear regression was used for continuous variables, including dietary B6 intake, supplemental B6 intake, alcohol consumption, systolic blood pressure, diastolic blood pressure, BMI, and age. The association of the categorical variable B6 status, using plasma PLP cutoffs for adequate B6 status, suboptimal B6 status, and B6 deficiency, with potentially influential categorical variables, including ethnicity, education level, total household income level, OC use, hormone therapy use, supplemental B6 use, and smoking, was tested using a Chi-square test. All analyses as described above were also conducted after stratification for supplemental B6 use. Confounding and collinearity were checked by dropping one variable each time and compared the changes of effect size (25,(2"…) and the standard errors of the effect size (DE 5,(DE"…). A confounder was identified as the effect size of any other variables changes > 10%. A collinearity was identified as the standard error of the effect size of any other variables changes > 10%. Backward elimination procedure was used to establish the best-fit multivariable regression model. Partial F test or Akaike’s Information Criterion was used to select the best model, depending on the types of models that were being compared.     96 4.3! Result 4.3.1! Participants Characteristics Two hundred and twenty-three women were recruited and eligible to be enrolled in the study. Completed C-DHQ IIs and demographic questionnaires were obtained from 223 participants and 222 participants, respectively. The mean BMI of these participants was at the upper limit of the normal range (i.e., 18.5–24.9 kg/m² [152,153]). The mean waist circumference was greater than 80 cm, which is the cut-off for an increased risk of metabolic complications in women [154] (Table 4-3). The majority of the participants reported to be of European ethnicity (75%), 14% of Chinese, 5% of South Asian, and the remaining 7% of “other” ethnicities including Northeast Asian, Southeast Asian, Latin American, and Arabian ethnicity. Over 91% of participants reported some post-secondary education and over 85% were in the upper-middle or highest bracket for total household income. Overall the sample was of high socio-economic status based on income and education indicators.     97 Table 4-3 General Participant Characteristics of Older Adult Women in Metro Vancouver Variables Distribution Total (n= 223) Age*, years Normal 60.4 ± 5.5 Weight†, kg Skewed 65.1 (63.2,"67.1) Height*, cm Normal 162.0 ± 6.8 Body mass index†, kg/m² Skewed 24.9 (24.2, 25.6) Waist circumference†, cm  Skewed 85.0 (83.2, 86.8) Hip circumference*, cm Normal 102.4 ± 11.4 Waist and hip ratio* Normal 0.84 ± 0.07 Systolic blood pressure*, mmHg Normal 120.5 ± 14.4 Diastolic blood pressure*, mmHg Normal 72.0 ± 9.7 Ethnicity‡  Total (n= 222) European  166 (75) Chinese  31 (14) South Asian  10 (5) || Other  15 (7) Education‡  Total (n= 222) Less than secondary school education  2 (0.9) Secondary school diploma  18 (8) Post-secondary education  78 (35) Bachelor’s degree  71 (32)  University degree or >than bachelor’s degree  53 (24) Total household income‡§  Total (n= 208) Lowest  4 (2) Lower-middle  27 (13) Upper-middle  64 (31) Highest  113 (54) * Values presented as arithmetic mean ± SD.  † Values presented as geometric mean (95% CI). ‡#Values presented as n (%). §Lowest: <$15001 if 1−2 people, <$20001 if 3−4 people, <$30001 if ≥5 people, Lower-middle: $15001−30000 if 1−2 people, $20001−40000 if 3−4 people, $30001−60000 if ≥5 people, Upper-middle: $30001−60000 if 1−2 people, $40001−80000 if 3−4 people, $60001−80000 if ≥5 people, Highest: >$60000 if 1−2 people, >$80000 if ≥3 people. || Northeast Asian, Southeast Asian, Latin American, and Arabian ethnicity  Most participants were in menopause (94%) and never used menopausal hormones (82%) (Table 4-4). About half of the participants never used OC (52%) whereas another half of the participants reported a history of OC use (48%). Nutritional supplement use was reported by 65% of the participants. Supplemental B6 use was reported by 30%. Most of the participants were either non-smoker (79%) or former smoker (18%).   98 Table 4-4 Lifestyle Characteristics of Older Adult Women in Metro Vancouver Variables*  Menopause Total (n= 222) No 14 (6) Yes 208 (94) Use of OC Total (n= 222) Never 115 (51.8) Former 106 (47.7) Current 1 (0.45) Use of hormone therapy Total (n= 222) Never 183 (82.4) Former 23 (10.4) Current 16 (7.2) Use of nutritional supplements Total (n= 223) No 78 (35) Yes 145 (65) Use of supplemental B6 Total (n= 223) No 157 (70) Yes 66 (30) Current or former smoker Total (n= 222) Never 176 (79.3) Former smoker 41 (18.5) Current occasional smoker (1–9 cigarettes/day) 3 (1.4) Current regular smoker (10–19 cigarettes/day) 1 (0.45) Current frequent smoker (≥20 cigarettes/day) 1 (0.45) *Values presented as n (%). Abbreviations: B6, vitamin B6; OC, oral contraceptives  99 4.3.2! Dietary Intakes Twelve samples were excluded due to plausible misreports in the C-DHQ; thus, a total of 211 samples were included in the dietary analysis. The mean total energy intake was 1454 (95% CI= 1385.6−1526.7) kcal/day (Table 4−5). The mean dietary B6 intake from food sources and mean total B6 intake from food and supplement were both above the EAR of B6 for older adult women (1.3 mg/day).  Table 4-5 Dietary Intake of Older Adult Women in Metro Vancouver Variables Distribution Total (n= 211) Total energy intake, kcal/day Skewed 1453.9 (1385.6, 1526.7) Dietary B6 intake from food sources, mg/day Normal 1.8 (1.7, 1.9) Total B6 intake from food and supplements, mg/day Skewed 3.3 (2.7, 3.8) Values presented as geometric mean (95% CI). Abbreviations: B6, vitamin B6  4.3.3! Primary Objective: prevalence of B6 deficiency and suboptimal status  Blood samples were obtained from 222 participants. There was one completely unsuccessful venipuncture. The distribution of plasma PLP concentrations was skewed to the left and thus log-transformed to perform all further analyses. Mean (95% CI) concentration of plasma PLP concentration was 85.6 (76.0, 96.5) nmol/L (Table 4-6). The prevalence of B6 deficiency and suboptimal B6 status was 1.4% and 8.1%, respectively.  Table 4-6 Plasma Pyridoxal 5’-phosphate Concentration and Prevalence of Vitamin B6 Deficiency, Suboptimal Status, and Adequacy in Older Adult Women in Metro Vancouver Variables Distribution Total (n= 222) Plasma PLP concentration†, nmol/L Skewed 85.6 (76.0, 96.5) Prevalence of B6 status categories‡   B6 deficiency (plasma PLP< 20 nmol/L)  3 (1.4) Suboptimal B6 status (plasma PLP= 20–30 nmol/L)  18 (8.1) Adequate B6 status (plasma PLP> 30 nmol/L)  201 (90.5) † Values presented as geometric mean (95% CI). ‡ Values presented as n (%). Abbreviations: B6, vitamin B6; PLP, pyridoxal 5’-phosphate   100 4.3.4! Secondary Objective: Predictors of Vitamin B6 Status 4.3.4.1! Bivariate Analysis Table 4-7 presents plasma PLP concentration by categories of demographic and lifestyle factors. Table 4-8 presents plasma PLP concentration by categories of demographic and lifestyle factors in non-users of supplemental B6, given that the use of supplemental B6 showed a high association with B6 status in the overall sample. The unadjusted associations between plasma PLP concentration and continuous variables are shown in Table 4-9.  Plasma PLP concentration significantly differed between users and non-users of supplemental B6; users of supplemental B6 had plasma PLP concentration of 222.7 nmol/L compared to 57.1 nmol/L in non-users of supplemental B6 (Table 4-7). Plasma PLP concentration was significantly inversely associated with BMI, systolic blood pressure, diastolic blood pressure, and positively with dietary B6 intake and supplemental B6 intake (Table 4-9). For every unit increase in BMI, plasma PLP concentration decreased by 4.3%. Both systolic and diastolic blood pressure were significantly associated with a decrease in plasma PLP concentration (−1.1% and −1.7%, respectively). In contrast, every milligram increase in dietary B6 intake was significantly associated with a 25.3% increase in plasma PLP concentration whereas every milligram of supplemental B6 intake was significantly associated with a 1.9% increase in plasma PLP concentration.  There was no significant difference in plasma PLP concentration based on ethnicity, education level, household income, OC use, hormone use or age in bivariate analyses. Similarly, there was no significant difference in the prevalence of B6 deficiency or suboptimal B6 status or both combined compared to adequate B6 status based on any demographic or lifestyle factors except that the prevalence of suboptimal B6 status was significantly higher in non-users of  101 supplemental B6. However, variables with borderline significance (P≤ 0.20) included ethnicity, OC use, hormone use, smoking, education level and total household income. Plasma PLP concentration was borderline different between ethnic groups (P= 0.17) and was borderline lower in users of OC (P= 0.17). Current or previous hormone users had a borderline higher prevalence of B6 deficiency (5.1%) compared to never users (0.5%) (P= 0.14). Women of low education level had a borderline higher prevalence of suboptimal B6 status (12.4%) compared to women of high income level (4.8%) (P= 0.07). Women of low income level had a borderline higher prevalence of suboptimal B6 status (16.1%) compared to women of high income level (5.7%) (P= 0.09). Current or previous smokers had a borderline higher prevalence of suboptimal B6 status (15.2%) compared to women of high income level (6.3%) (P= 0.10).  The full model with plasma PLP concentration as the dependent variable controlled for dietary B6 intake, supplemental B6 intake, ethnicity, OC use, hormone use, BMI, systolic blood pressure, diastolic blood pressure, smoking, education level and total household income status.  102 Table 4-7 Plasma Pyridoxal 5’-phosphate Concentration by Categories of Demographic and Lifestyle Factors in Older Adult Women in Metro Vancouver Variables Participants, n Mean (95% CI), nmol/L P-value <20 nmol/L, n (%) P-value 20–30 nmol/L, n (%) P-value Overall 222 85.6 (76.0, 96.5)  3 (1.4)  18 (8.1)  Ethnicity        European* 166 82.7 (72.1, 94.8) 0.17 3 (1.8) 0.80 14 (8.4) 0.16 Chinese 31 105.4 (76.3, 145.7)  0  1 (3.2)  South Asian 10 123.8 (67.5, 227.3)  0  0  Other† 14 64.5 (43.4, 96.2)  0  3 (21.4)  Education            High* 124 90.1 (77.2, 105.1) 0.36 2 (1.6) 1 6 (4.8) 0.07 Low 97 80.6 (66.9, 97.1)  1 (1.0)  12 (12.4)  Household income        High* 176 86.9 (76.6, 98.7) 0.46 2 (11.4) 0.93 10 (5.7) 0.09 Low 31 76.4 (57.8, 101.0)  1 (3.2)  5 (16.1)  Current or previous OC use        No* 115 92.9 (78.7, 109.7) 0.17 0 0.22 8 (7.0) 0.67 Yes 106 78.6 (66.3, 93.3)  3 (2.8)  10 (9.4)  Current or previous hormone use        No* 182 87.9 (76.9, 100.4) 0.39 1 (0.5) 0.14 15 (8.2) 1 Yes 39 76.6 (59.1, 99.4)  2 (5.1)  3 (7.7)  Supplemental B6 use        No* 156 57.1 (51.6, 63.3) <0.001 3 (1.9) 0.62 18 (11.5) 0.009 Yes 66 222.7 (190.7, 260.1)  0  0  Current or previous smoker        No* 175 87.4 (76.7, 99.4) 0.56 2(1.1) 1 11(6.3) 0.10 Yes 46 80.2 (60.0, 107.1)  1(2.2)  7(15.2)  *Referent category. Difference in plasma pyridoxal 5’-phosphate concentration between groups was assessed for dichotomous variables by two sample t-test and for categorical variables by one-way ANOVA. Difference in prevalence of plasma pyridoxal 5’-phosphate concentration < 20 nmol/L and 20–30 nmol/L was assessed by chi-square test.  † Northeast Asian, Southeast Asian, Latin American, and Arabian ethnicity Abbreviations: B6, vitamin B6; OC, oral contraceptive  103 Table 4-8 Plasma Pyridoxal 5’-phosphate Concentration by Categories of Demographic and Lifestyle Factors in Non-users of Supplemental Vitamin B6 in Older Adult Women in Metro Vancouver Variables Participants, n Mean (95% CI), nmol/L P-value <20 nmol/L, n (%) P-value 20–30 nmol/L, n (%) P-value Overall 156 57.1 (51.6, 63.3)  3 (1.9)  18 (11.5)  Ethnicity        European* 118 56.9 (50.5, 64.3) 0.69 3 (2.5) 0.81 14 (11.9) 0.30 Chinese 19 58.2 (47.1, 72.0)  0  1 (5.3)  South Asian 6 74.1 (35.9, 152.8)  0  0  Other† 12 50.4 (38.2, 66.3)  0  3 (25)  Education            High* 83 60.7 (52.7, 70.0) 0.21 2 (2.4) 1 6 (7.2) 0.11 Low 72 53.3 (46.0, 61.7)  1 (1.4)  12 (16.7)  Household income        High* 122 57.9 (52.0, 64.2) 0.56 2 (1.6) 0.99 10 (8.2) 0.13 Low 24 53.3 (41.4, 68.6)  1 (4.2)  5 (20.8)  Current or previous OC use        No* 76 57.3 (50.6, 65.0) 0.95 0 0.26 8 (10.5) 0.87 Yes 79 57.0 (48.5, 66.8)  3 (3.8)  10 (12.7)  Current or previous hormone use        No* 129 59.1 (52.7, 66.3) 0.14 1 (0.7) 0.12 15 (11.6) 1 Yes 26 48.3 (39.5, 59.1)  2 (7.7)  3 (11.5)  Current or previous smoker        No* 124 58.0 (52.3, 64.4) 0.56 2 (1.6) 1 11 (8.9) 0.07 Yes 31 53.8 (40.1, 72.2)  1 (3.2)  7 (22.6)  *Referent category. Difference in plasma pyridoxal 5’-phosphate concentration between groups was assessed for dichotomous variables by two sample t-test and for categorical variables by one-way ANOVA. Difference in prevalence of plasma pyridoxal 5’-phosphate concentration < 20 nmol/L and 20–30 nmol/L was assessed by chi-square test. † Northeast Asian, Southeast Asian, Latin American, and Arabian ethnicity Abbreviations: CI, confidence interval; OC, oral contraceptive  104 Table 4-9 Unadjusted Association of Continuous Variables with Plasma Pyridoxal 5’-phosphate Concentration in Older Adult Women in Metro Vancouver Variables Participants, n Unadjusted % change in plasma PLP (95% CI)* P-value Age, years 222 0.3 (−1.8,#2.5) 0.79 BMI, kg/m²  222 −4.3 (−6.2, −2.4) <0.001 Systolic blood pressure, mmHg 222 −1.1 (−1.9, −0.3) 0.008 Diastolic blood pressure, mmHg 222 −1.7 (−2.9, −0.6) 0.004 Dietary B6 intake, mg/day 211 25.3 (9.3,#43.7) 0.001 Supplemental B6 intake, mg/day 211 1.9 (1.5,#2.4) <0.001 Alcohol intake, g/day 211 0.3 (−1.0, 1.6) 0.67 * The percentage change in plasma PLP concentration was calculated by (!"# − 1)*100% determined by simple linear regression. Abbreviations: B6, vitamin B6; BMI, body mass index; PLP, pyridoxal 5’-phosphate   4.3.4.2! Multivariable Regression Model Dietary B6 intake, supplemental B6 intake, and South Asian ethnicity were positively associated with plasma PLP concentration; BMI was inversely associated with plasma PLP concentration (Table 4-10). The model explained 33% (adjusted R²) of the variance in plasma PLP concentration.  Compared to South Asian ethnicity, Chinese ethnicity and other ethnicity did not significantly associate with the change in plasma PLP concentration. However, South Asian ethnicity was associated with a 65% increase in plasma PLP concentration adjusting for BMI, dietary and supplemental B6 intake (P= 0.048). In the adjusted model, every unit increase in BMI was estimated to decrease plasma PLP concentration by 3.5%; every milligram increase in dietary B6 intake and supplemental B6 intake was associated with a 13.9% and 1.8% increase in plasma PLP concentration, respectively.    105 Table 4-10 Predictors of Plasma Pyridoxal 5’-phosphate Concentration in Older Adult Women in Metro Vancouver Variables Unadjusted  % change in plasma PLP (95% CI)* Unadjusted P-value Adjusted  % change in plasma PLP (95% CI)* Adjusted P-value Ethnicity†       Chinese ethnicity 21.7 (−15.1, 74.4) 0.28 −6.3 (−31.0, 27.3) 0.68   South Asian ethnicity 32.8 (−27.3, 142.5) 0.35 65.0 (0.5, 170.8) 0.048   Other ethnicity‡ −19.8 (−52.6, 35.8) 0.41 −16.0 (−45.5, 29.4) 0.43 BMI, kg/m² −4.3 (−6.2, −6.4) <0.001 –3.5 (–5.2, –1.7) <0.001 Dietary B6 intake, mg/day 26.1 (10.1,#44.5) <0.001 13.9 (1.6, 28.2) 0.03 Supplemental B6 intake,    mg/day 1.9 (1.5,#2.3) <0.001 1.8 (1.3, 2.2) <0.001 Number of observation = 209, Model P <0.001, R²: 0.35, Adjusted R²: 0.33.  * The percentage change in plasma PLP concentration was calculated by (!"# − 1)*100%. † Referent category: European ethnicity ‡ Northeast Asian, Southeast Asian, Latin American, and Arabian ethnicity Abbreviations: B6, vitamin B6; BMI, body mass index; PLP, pyridoxal 5’-phosphate   4.4! Discussion In this study, a 1.4% prevalence of B6 deficiency and an 8.1% prevalence of suboptimal B6 status was identified in older adult women in Metro Vancouver. The results are similar to the findings in young adult women, which revealed a 12.4% combined prevalence of B6 deficiency and suboptimal B6 status. 4.4.1! Prevalence of Vitamin B6 Deficiency and Suboptimal Vitamin B6 Status The mean plasma PLP concentration was 85.6 nmol/L in these older adult women who self-reported no major health conditions. In the CCHS in 2004, a 19.4% prevalence of inadequate dietary B6 intake was reported among older adult women (51−70 years) compared to a 9.6% prevalence in young adult women (19−30 years) [20]. However, this did not reflect on the biochemical B6 status as the prevalence of B6 deficiency and suboptimal B6 status was similar between young and older adult women in this thesis. In NHANES 2003−2004, adults aged 21−44 years were reported to have particularly low B6 status compared to other age groups even  106 though teenagers had the lowest B6 intakes [22]. Morris et al. [22] suggested that estrogen plays an important role in explaining the specifically low plasma PLP concentration in young adult women. In the NHANES cohort with women across all ages, plasma PLP concentration was shown to decrease from menarche age to age of 20−40 years and gradually returns at menopause age [22]. The high prevalence of B6 deficiency found in OC users also indicates the influence of estrogen in women [22].Our result was consistent with another Canadian cross-sectional study in pre- and postmenopausal women which showed that the mean plasma PLP concentration in both respective groups was above the cut-off for adequate B6 status [118]. However, neither the mean plasma PLP concentration nor the prevalence of B6 deficiency and suboptimal B6 status was reported in that study [118]. In the study of Ye et al. [25], a cross-sectional study in Boston reported 10.7% of B6 deficiency and 17.8% of suboptimal B6 status among Puerto Rican women aged 45−75 years  (n= 889). The lower prevalence seen in our study might be due to the different participant characteristics. The participants in the Ye et al. study were recruited for a longitudinal study focusing on relationships between stress, nutrition, and chronic health conditions and they had lower socioeconomic status (48% had education lower than grade 9th and 29% lived in poverty) as compared to this study [25]. It is not surprising that a lower prevalence of suboptimal B6 status was reported in this study since B6 status has been inversely associated with income level [24,25] and the risk of cardiovascular disease [4–9], colorectal cancer [10,11,106], breast cancer [12,13], lung cancer [14,107] and ovarian cancer [140]. The 1.4% prevalence of B6 deficiency in this sample was much lower than the 26% reported for adults aged >65 years in the NHANES 2003−2004 [22]. This large discrepancy is likely due to the high socioeconomic status of the women in this study and the different  107 analytical methods of plasma PLP quantification compared with the NHANES data, as discussed in details in section 3.4.1. In another population-based study in Italy, a median of 25.2 nmol/L serum PLP concentration among older adults (median age= 69 years) was much lower than the mean plasma PLP concentration in this study [130]. The prevalence of B6 deficiency or suboptimal B6 status was not reported [130]. The older population (78.8% of participants >65 years) in that study may explain the lower PLP concentration as age was negatively associated with serum PLP concentration [130]. The limited data on biochemical B6 status in Canadians [20,21,131] and the 9.5% prevalence of low B6 status in a cohort of healthy older adult women of high socioeconomic status indicates the need for a representative, population-based study to determine the B6 status of Canadians. A higher prevalence of B6 deficiency and suboptimal B6 status might be observed in a representative sample of Canadian women compared to this study. 4.4.2! Predictors of Plasma Pyridoxal 5’-phosphate Concentration 4.4.2.1! Supplemental Vitamin B6 Intake The use of supplemental B6 was a strong predictor of plasma PLP concentration both before and after multivariable adjustment in this study. In this sample, 65% of women used general nutrition supplements and 30% of all participants (i.e., 46% of general nutrition supplement users) used B6-containing supplements. The prevalence of nutritional supplement use in this sample was very similar to the prevalence reported in the CCHS in 2004 [141] which 61% of Canadian women aged 51−70 years reporting nutritional supplement use, though the prevalence of supplemental B6 use was not reported. In this study, the combined prevalence of B6 deficiency and suboptimal B6 status was 13.5% in non-users of supplemental B6 and zero in supplemental B6 users. This is consistent with the US NHANES data that reported higher plasma  108 PLP concentrations and a lower prevalence of B6 deficiency in general nutrition supplement users [22] and supplemental B6 users [23] compared with non-users. Since the prevalence of supplemental B6 use was not reported in the CCHS data and the majority of women, as seen in this study, did not use B6 containing supplements, a prevalence of B6 deficiency and suboptimal B6 status higher than 9.5% can be expected in general Canadian women.  4.4.2.2! Dietary Vitamin B6 Intake Similar to other studies [22,25], a strong association between total B6 intake and plasma PLP was also observed. The C-DHQ II used in this study is the only FFQ that uses CCHS data to develop and evaluate the food list, portion size categories and nutrient database [146]. However, the mean dietary B6 intake from this study was incomparable with the dietary B6 intakes reported in the CCHS in 2004 given the different tool they used (24-hr recalls) to assess the mean dietary B6 intake. The mean total energy intake of 1454 kcal/day in this study was lower than the 1668 kcal/day reported in British Columbia women aged 51−70 years in CCHS 2004, which suggests that there might be under-reporting for total energy and dietary B6 intake in this study. Although the C-DHQ II was designed to assess dietary intakes in the Canadian population [146], 25% of study participants were of an ethnicity other than European and thus it is possible that the C-DHQ II was unable to record certain ethnic-specific foods. Research has shown that substantial numbers of food items were missed in Canadian designed FFQ when assessing dietary intake of Chinese and South Asians [155]. Ethnic-specific FFQs have 61 (37%) and 55 (33%) questions designed specifically for South Asian and Chinese, respectively [155]. 4.4.2.3! Ethnicity South Asian ethnicity was not associated with plasma PLP in the unadjusted model; however, this association became significant after adjustment for dietary B6 intake, supplemental  109 B6 intake, and BMI. The positive association between South Asian ethnicity and plasma PLP was not expected since an inverse association after adjustment for BMI, relative dietary B6 intake, and supplemental B6 use was observed in the sample of young adult women described in Chapter 3. Both studies have shown that the difference in B6 status between South Asian and European women was not related to dietary B6 intake or supplemental B6 use. However, this finding should be interpreted with caution since neither the C-DHQ II used in this study nor the semi-quantitative FFQ used in young adult women study was designed for specific ethnicity and may fail to report some foods (such as chapati and chicken curry are high in B6 or channa dhal that is low in B6 [156]) in South Asian diet. In addition, both studies had a small sample size of South Asian women (n= 56 in young adult women and n=10 in this study) and could not represent the South Asian population in Vancouver. Ethnicity has been correlated with nutritional biomarker levels of other micronutrients (e.g. ferritin, folate) in NHANES 1999−2002 [142]. The discrepancy seen in the young adult women study and this study suggests that additional research with a larger sample size of South Asians is needed to confirm the association between South Asian and B6 status. 4.4.2.4! Body Mass Index BMI was inversely associated with plasma PLP after adjustment for dietary B6 intake, supplemental B6 intake, and ethnicity. The inverse relationship between BMI and plasma PLP was consistent with the result of the NHANES data where every 25% increase in BMI was associated with a 13% decrease in plasma PLP [24]. The inverse linkage between body mass and B6 status has been suggested to the result of volumetric dilution [89]. Furthermore, plasma PLP has been inversely associated with circulating concentration of C-reactive protein, a marker of systemic inflammation that is elevated in obesity [76,90]. Inflammation has been suggested to  110 induce B6 deficiency by increasing the utilization of B6 in sites of inflammation, leading to the redistribution of B6 and low circulating PLP concentrations [112,113,116]. In a longitudinal study in older Scottish women, BMI significantly increased over the 20-year follow-up period and 72% of women gained >5% of their baseline BMI [157]. Postmenopausal BMI has been associated with estrogen metabolism which has been linked with a higher breast cancer risk [158]. Since low B6 status has also been associated with an increased risk of postmenopausal breast cancer [12,13], it would be crucial for further research to understand the role of B6 in the BMI-breast cancer association in postmenopausal women.  4.4.2.5! Other Variables In this study, 94% of women were in menopausal status and 51% of them reported past OC use when they were menstruating. Compared to the NHANES data, where 25% of B6 deficiency was reported in menopausal women aged ≥ 45 years who were past OC users [22], we did not find past OC use to be a significant predictor of B6 status. Current evidence suggested that estrogen content in OCs disrupt tryptophan metabolism independent of B6 status [67]. Hormone therapy has shown to affect plasma PLP concentration [69]; however, hormone use was not a significant predictor of plasma PLP after multivariable adjustment in this study. The sample size of current and former hormone users in this study (n= 26) may be too small to detect a statistically significant effect of hormone use on plasma PLP concentration. This sample also lacked the power to detect associations with other variables, such as smoking (only 46 participants were former or current smokers).  Plasma PLP was inversely associated with both systolic and diastolic blood pressure in simple linear regression models but the association attenuated after adjustment for ethnicity, BMI, dietary B6 intake, and supplemental intake. BMI was found to be positively correlated with  111 systolic (r= 0.41, P< 0.001) and diastolic blood pressure (r= 0.32, P< 0.001). The attenuation after multivariable adjustment suggests that the inverse relationship between plasma PLP and blood pressure might be explained by BMI. Higher BMI has been positively associated with hypertension and mean arterial pressure in older adults aged ≥ 50 years in a large multi-continent study (n= 52,946) [159]. Current evidence has inconclusive results for the association between plasma PLP and blood pressure [160,161]. However, since low B6 status has been associated with cardiovascular disease [4–9] and hypertension is a risk factor for cardiovascular disease, more research is needed to confirm the association between B6 status and blood pressure. Riboflavin and B6 are interrelated as pyridoxamine 5’-phosphate reductase requires riboflavin formed flavin mononucleotide as a cofactor in the conversion of B6 vitamers [162]. Moreover, B6 and riboflavin are both involved in one-carbon metabolism where riboflavin serves as a coenzyme for methylenetetrahydrofolate reductase. Riboflavin status and the methylenetetrahydrofolate reductase gene polymorphism are key variables to investigate in these women since riboflavin supplementation has been found to reduce blood pressure in patients with the methylenetetrahydrofolate reductase gene mutation [163,164] and this polymorphism has been associated with an increased risk of hypertension [165,166].  4.4.3! Strengths and Limitations The strength of this study is that it is the first study to report on the biochemical B6 status in older adult women in Canada. However, there are a number of limitations to this study. First, the recruitment of a convenience sample leading to the enrolment of relatively healthy women of high socioeconomic status. Over 56% of our study participants obtained at least a bachelor’s degree, which was higher than the reported 35% among women aged 25−64 years in Metro Vancouver based on results from the National Household Survey in 2011 [143]. Therefore, the  112 results of this study cannot be extrapolated and are not representative for all Canadian older adult women. Yet studies have found that B6 status differed between income levels but not education levels [24,25]. The higher socioeconomic status in this study compared to the overall population of Canadian women limits the generalizability of the findings of this study.  Second, while the use of C-DHQ II is able to assess habitual intakes in a relatively simple, cost-effective, and time-efficient manner, it is a less preferable tool to assess dietary intakes compared with the 24-hour recall that records detailed food consumption in the past 24 hours. In addition, although the older version of the US DHQ I has been validated to measure nutrient intakes in adults with 24-hour recall [147,148] and there was some modification made in the C-DHQ II to reflect the Canadian food supply, such as the inclusion of ethnic foods and expansion of the number of questions on dietary supplements, C-DHQ II has not yet been validated. It is still unclear whether it is a suitable tool to assess micronutrient intakes. Thus, the findings on dietary B6 intake should be interpreted with caution since C-DHQ II might not adequately capture the dietary B6 intake of the participants in this study. Third, the four variables in the multiple linear regression model only explained 33% of the variability in plasma PLP concentration. The results might be confounded by unexplained biological factors, such as physical activity, inflammation status and genetic variants. The positive association between B6 status and physical activity levels has been identified in two large population-based studies [24,25]. C-reactive protein, an inflammation marker, has been associated with B6 status independent of dietary B6 intake [76,114,115]. Variants in tissue-nonspecific alkaline phosphatase gene have been shown to influence plasma PLP concentration, but whether it indicates a functional change in B6 status remains unclear [144]. In addition, the  113 small sample size of this study likely had not enough power to detect associations with other variables including hormone use, and smoking. Another limitation of this study was the use of plasma PLP as the single biomarker to assess B6 status. Plasma PLP was chosen because it is currently the only indicator with established cut-offs to define B6 deficiency and B6 adequacy [2]. However, plasma PLP only reflects the circulating concentration of B6 and is influenced by several factors, such as inflammation, serum albumin, and alkaline phosphatase concentrations [38]. The lack of information on inflammation status in this study weakens the validity of using plasma PLP as the single indicator for B6 status. Functional indicators, for example, plasma cystathionine [48,51,52] and plasma kynurenines [38,41,42] has been found to reflect intracellular B6 status. Evaluating these functional indicators would give a complete picture of biochemical B6 status.   4.5! Conclusion A 9.5% combined prevalence of B6 deficiency and suboptimal B6 status was found in this convenience sample of 222 healthy older adult women in Metro Vancouver. B6 adequacy is crucial for life-long health and women with higher BMI are more likely to have lower B6 status. More research is needed to confirm the relationship between South Asian ethnicity and B6 status. Increasing dietary B6 intake or taking B6-containing supplements may help increase women’s biochemical B6 status. The lower prevalence of B6 deficiency and suboptimal B6 status in these older adult women compared to the reports from representative samples in other countries may be due to the high socioeconomic status in this study. Since low B6 status has been associated with an increased risk of cardiovascular disease and cancers, evaluating B6 status in a representative sample of Canadian women is necessary.  114 Chapter 5:!General Discussion and Conclusion 5.1! Summary Vitamin B6, in the form of PLP, plays an essential role in the metabolism of amino acids, synthesis of neurotransmitters, regulation of energy homeostasis, and metabolism of heme. Adequate B6 status is important for women in different life stages. Low preconceptional B6 status has been associated with an increased risk of preterm birth and early miscarriage [15], and a reduced probability of conception [16]. Women of childbearing age should maintain an adequate B6 status to ensure healthy pregnancy outcomes. Low B6 status has been associated with cardiovascular disease [4–9] and several types of cancers [10–14]. Specifically, high circulating B6 concentration has been associated with a reduced risk of postmenopausal breast cancer [12,13]. In the US, a high prevalence of B6 deficiency was found in adult women in NHANES 2003−2004 [22]. In Canada, the CCHS 2004 reported that 19% of adult women had an inadequate dietary B6 intake [20]. However, there is limited data available on biochemical B6 status in Canadian women. The results of this thesis have consistently shown a 2% prevalence of B6 deficiency and an about 10% prevalence of suboptimal B6 status in both young adult women (Chapter 3) and older adult women (Chapter 4) in Metro Vancouver. The predictors of plasma PLP identified in this thesis are dietary B6 intake, supplemental B6 use, South Asian ethnicity, and BMI. This chapter begins with a summary of the sample characteristics, methods, and outcomes for Chapters 3 and 4 (Table 5-1). This is followed by a general discussion of the results, including study limitations, and concludes with future directions of the research.  115 Table 5-1 Summary of Vitamin B6 Status in Young and Older Adult Women in Metro Vancouver  Chapter 3: Young adult women Chapter 4: Older adult women Sample size, n 202 223 Sampling method Convenience sampling Convenience sampling Age range, years 19−35 51−70 Ethnicity 73% European, 27% South Asian 75% European, 14% Chinese, 5% South Asian, 7% other BMI 22.7 (22.2, 23.1) 24.9 (24.2, 25.6) Education level 71% Bachelor’s degree or higher 56% Bachelor’s degree or higher Income level 56% high income level 85% high income level Supplemental B6 use, % 28 30 Dietary assessment Semi-quantitative FFQ C-DHQ II Plasma PLP quantification HPLC with fluorescence detection LC/MS-MS Variables included in analysis Height, weight, age, waist circumference body mass index, ethnicity, physical activity, OC use, nutritional supplement use, supplemental B6 use, smoking, income level, education level, anemia, plasma PLP concentration, energy intake, protein intake, dietary B6 intake Height, weight, age, waist circumference body mass index, blood pressure, ethnicity, OC use, hormone use, nutritional supplement use, supplemental B6 intake, smoking, income level, education level, anemia, plasma PLP concentration, energy intake, protein intake, dietary B6 intake Plasma PLP concentration, nmol/L 61.0 (55.2, 67.3) 85.6 (76.0, 96.5) Prevalence of B6 deficiency, % 1.5 1.4 Prevalence of suboptimal B6 status, % 10.9 8.1 Predictors of plasma PLP Dietary B6 intake, supplemental B6 use,  South Asian ethnicity, BMI Dietary B6 intake, supplemental B6 intake, South Asian ethnicity, BMI Abbreviations: BMI, body mass index; C-DHQ, Canadian Diet History Questionnaire; FFQ, food frequency questionnaire; HPLC, high performance liquid chromatography; LC/MS-MS, liquid chromatography-tandem mass spectrometry ; OC, oral contraceptive; PLP, pyridoxal 5’-phosphate  116 5.2! General Discussion 5.2.1! Vitamin B6 Status in Young and Older Adult Women in Metro Vancouver We found similar prevalence of B6 deficiency and prevalence of suboptimal B6 status between samples of young and older adult women. In the CCHS 2004, an 18% prevalence of inadequate dietary B6 intake was reported in adult women with higher prevalence among older adult women aged 51−70 years (19.4%) compared to young adult women aged 19−30 years (9.6%) [20].  Our results did not confirm the hypothesis that a higher prevalence of B6 deficiency and suboptimal B6 status would be seen in older adult women given the higher prevalence of inadequate B6 intake in the CCHS 2004. Supplemental B6 use was a strong predictor of plasma PLP concentration in both samples. The similar prevalence of supplemental B6 use among young and older adult women (28% and 30%, respectively) might partially explain the analogous prevalence of B6 deficiency and suboptimal B6 status.  The mean BMI in young adult women was 22.7 kg/m2 whereas it was 24.9 kg/m2 in older adult women. BMI was inversely associated with plasma PLP concentration in both samples of young and older adult women. Yet, this did not reflect on their mean plasma PLP concentration. The mean plasma PLP concentration was higher in older adult women than young adult women (85.6 versus 61 nmol/L, respectively). The inconsistency is likely due to the small association between plasma PLP and BMI. For every unit increase in BMI, a 3.5% and 2.7% decrease in plasma PLP concentration was observed in the sample of older and young adult women, respectively. In addition, plasma PLP concentration of women has been reported to be influenced by age, independent of their supplemental B6 use in NHANES 2003−2004 [22]. Women appear to have their lowest plasma PLP concentration during the young adult years (e.g. 21−44 years) as seen in both users and non-users of supplemental B6 [22]. This age pattern starts from the  117 menarche age and gradually ends when women enter menopausal age [22], which suggested that estrogen might play an important role in explaining plasma PLP concentration in women. Furthermore, the high prevalence of B6 deficiency in OC users also indicates the influence of estrogen in women [22].   South Asian ethnicity was identified as a predictor of plasma PLP concentration in this thesis but with conflicted associations between the samples of young and older adult women. Women of South Asian ethnicity were estimated to have 21% lower plasma PLP concentration compared to women of European ethnicity after adjustment for BMI, dietary B6 intake, and supplemental B6 use in the sample of young adult women. In contrast, older adult women of South Asian ethnicity were expected to have 65% higher plasma PLP concentration compared to women of European women. These results should be interpreted with caution since we had a small sample size of South Asian women in both studies (n= 56 and 10, respectively). A study in Ottawa has found that South Asians had significantly lower vitamin D status and higher risk factors for developing cardio-metabolic diseases compared to European [167]. In addition, a study investigated the differences in biomarker concentrations in populations across continents has shown that the Chinese population have much higher prevalence of B6 deficiency compared to the US population (23.5% versus 9.4%) [168]. The differences in body fat and BMI has been reported between Asian and European where Asians have 3−5% higher body fat compared to European while having the same BMI [169]. This suggests that body fat percentage might be a better marker to account for the difference in body composition between South Asian and European when assessing the association between ethnicity and B6 status. South Asian ethnicity might not be a predictor of B6 status if multivariable regression models adjusted for body fat percentage rather than BMI. Given that our studies are the first to report an association between  118 B6 status and South Asian ethnicity, more research is required to elucidate the direction of the association and the potential underlying mechanisms.  5.2.2! Limitations The cross-sectional design of the two studies in this thesis limits the ability to draw causal relationships about any of the observed results. In addition, the use of convenience sampling method in both of the studies might cause selection bias in these participants because individuals who volunteer to participate in research studies tend to have higher socioeconomic status and be more health conscious compared with the general population [170,171]. The convenience samples of the studies presented in this thesis are thus not representative of young and older adult women in Metro Vancouver or Canada. The overall small sample size and the small number of women who reported OC use, hormone use, and/or smoking limited the ability to detect any significant associations of these variables with plasma PLP concentration. Although we identified South Asian ethnicity to be a predictor of plasma PLP concentration, the inconsistent results and the small sample size in both studies suggest the need for further investigation. Another limitation of this thesis is the use of FFQs as the dietary assessment tool. Both the semi-quantitative FFQ used in the young adult women study and the C-DHQ II used in the older adult women study were designed to look at the past year’s intake. This timeframe may pose a risk for increased recall bias, especially for season specific intake. The dietary intakes reported in these studies only provide an estimation of usual intake and may not reflect short-term dietary intakes or dietary changes. In addition, the questionnaires were neither designed nor validated for specific ethnic groups. Although we found South Asian ethnicity is a significant  119 predictor of plasma PLP concentration independent of dietary B6 intake in both studies, it is possible that the FFQs were unable to properly reflect the dietary intake of South Asian women. Although plasma PLP is the most commonly used B6 biomarker and the only indicator that has established cut-offs for adequate and deficient B6 status, it is not ideal to use one single indicator to assess biochemical B6 status. Describing B6 status solely with plasma PLP limits the generalization of the findings in healthy populations since plasma PLP has been associated with inflammation status, serum albumin, and alkaline phosphatase concentrations [38]. In addition, while plasma PLP indicates circulating B6 status, functional indicators reflect the metabolic status of PLP-dependent enzymes or pathways. Assessing B6 status without functional indicators does not allow a comprehensive evaluation of one’s B6 status.  5.2.3! Future Directions There are several suggestions for future studies to build upon this research and to provide further understanding about B6 status in Canadian women. First, since there has been no representative data on biochemical B6 status in Canada and that the present studies were comprised of healthy, high socioeconomic status women, the investigation of B6 status in a broader sample of women in Canada is needed with adequate representation from all demographic and lifestyle groups. Secondly, future studies should assess the contributions of other potential risk factors observed to affect B6 status, such as OC use, hormone use, smoking, alcohol consumption, genetic variants, inflammation status, and physical activity, using appropriately sensitive tools given that these associations have been made in other countries. Furthermore, assessment of dietary B6 using a validated FFQ along with 24-hour recalls will better capture dietary intakes including usual and current dietary intake. Since 21% of Canadian population are immigrants [81], ethnic-specific questions or questionnaires are crucial to assess  120 the ethnically diverse diets in all Canadians. Lastly, evaluation of B6 status with functional indicators will be important to establish reference values and ultimately aid the comprehensive assessment of suboptimal B6 status and B6 deficiency.  5.3! Conclusion Through the work conducted for this thesis, we are the first to report biochemical B6 status in healthy, non-pregnant, young adult women and older adult women in Canada. We have observed that based on plasma PLP concentration, which is the only indicator with established cut-offs, low B6 status was common in healthy, highly educated, young adult women aged 19−35 years and older adult women aged 51−70 years in Metro Vancouver. Dietary B6 intake, supplemental B6 intake, South Asian ethnicity, and BMI were the significant predictors of plasma PLP concentration. However, due to the inconsistent associations between plasma PLP and South Asian ethnicity, and the small sample size of South Asian women in this thesis, the results should be interpreted with caution. The findings in this thesis show concern regarding low B6 status of women in this region and potentially elsewhere in Canada, though they cannot be generalized to all women aged 19−35 years and 51−70 years in Metro Vancouver. Since low B6 status has been associated with an increased risk of poor pregnancy outcomes, cardiovascular disease, and cancer, women should maintain an adequate B6 status throughout the adult years. Potential at risk populations, such as women with inadequate dietary B6 intake, not using B6 containing supplements, having elevated BMI, and young women of South Asian ethnicity, should be adequately screened for vulnerability to B6 deficiency. It is also important that future studies assess B6 status with functional indicators such that reference values for multiple indicators will be able to build and  121 ultimately improve our current assessment for B6 deficiency. This thesis will hopefully instigate representative research on the biochemical B6 status among Canadian women.  122 Bibliography 1. Ho, C.-L.; Quay, T.; Devlin, A.; Lamers, Y. Prevalence and Predictors of Low Vitamin B6 Status in Healthy Young Adult Women in Metro Vancouver. Nutrients 2016, 8, 538. 2. 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Nat Rev Cancer 2016, 16, 650–662.                         145  Appendices Appendix A   Screening Questionnaire ADEQUACY OF VITAMIN B6 STATUS IN OLDER ADULT WOMEN IN METRO VANCOUVER – SCREENING QUESTIONNAIRE  – BY TELEPHONE  Subject ID # __ __ __ __ (####)* Please repeat this field at the top right corner of every separate page *Please note that all information on this questionnaire is labelled with an ID number only, and stored separately from your name and contact details. All information collected will be stored securely, and personally identifiable information will be accessible to no one except Dr Yvonne Lamers and her research associates. !Today’s Date (DD/MM/YYYY) ___ ___ /___ ___ /___ ___ ___ ___ !PERSONAL!INFORMATION! WHAT IS YOUR AGE? ___ ___ (years) (Exclude if not 51-70 yr)  HEALTH!and!PHARMACEUTICAL!USE! DO YOU SUFFER FROM A CHRONIC CONDITION   (For Example: Diabetes , Cardiovascular Disease , Cancer , Renal Failure/Kidney Disease, Chronic Obstructive Pulmonary Disease (COPD), Emphysema, Bronchitis, Arthritis, Fibromyalgia, Asthma, Stroke , Thyroid Condition, Bipolar disorder, Schizophrenia, Anxiety Disorder,  Learning Disability (Attention Deficit Disorder, Attention Deficit Hyperactivity Disorder, Dyslexia, Other), Eating Disorder (Anorexia, Bulimia etc.), Liver Disease, Gallbladder Problems, Hepatitis, HIV/AIDS)  Yes (01)  No (02) (Exclude if Yes)                         146   CURRENTLY (IN THE PAST MONTH) DID YOU TAKE ANY PRESCRIPTION MEDICATIONS?  (In addition to other prescription medication this includes: insulin, nicotine patches) *Do not indicate yes if you ONLY receive a prescription for hormonal contraceptives or postmenopausal hormone therapy   Yes (01)        No (02)  IF YOU ANSWERED YES TO THE LAST QUESTION, DO YOU TAKE ANY OF THE FOLLOWING MEDICATIONS? Hydralazine (Apresoline), Phenelizine (Nardil), Tranylcypromine (Parnate), Amiodarone (Cordarone), Phenobarbital (Luminal), Phenytoin (Dilantin), Levodopa, Metformin, Anti-Cancer Treatment, Antibiotics, Proton Pump Inhibitors, Antacids, Chloramphenicol   Yes (01)        No (02) (Exclude if Yes)  IF THE MEDICATION YOU TAKE IS NOT ONE OF THE PREVIOUS PLEASE SPECIFY BELOW:  ……………………………………………………………..  (Exclude if a medication is being taken for chronic illness or if B6 interaction exists)  CURRENTLY (IN THE PAST MONTH) DID YOU TAKE ANY PERFORMANCE ENHANCING OR RECREATIONAL DRUGS? (such as steroids, marijuana, cocaine etc)  Yes (01)        No (02) (Exclude if yes)  DO YOU SUFFER FROM ANY OF THE FOLLOWING GASTROINTESTINAL CONDITIONS? Atrophic Gastritis, Crohn’s Disease, Irritable Bowel Syndrome, Colitis, Pernicious Anaemia Celiac Disease, Acid Indigestion, Diverticulitis/Diverticulosis, Celiac Disease,?  Yes (01)        No (02) (Exclude if yes)    HAVE YOU UNDERGONE ANY FORM OF GASTRIC BYPASS SURGERY?  Yes (01)        No (02) (Exclude if yes)  Thank you for your time. If you meet the criteria for subject recruitment we will be in contact within 3-6 business days. If you do not hear from us, we apologize but we cannot include you in the study.                        147  Appendix B  Study Flyer B.1!                          148  B.2!                          149  B.3!                            150   Appendix C  Study Brochure C.1!                            151  C.2!                           152   Appendix D  Informed Consent Form    Subject Information and Consent Form  Adequacy of Vitamin B6 Status in Older Adult Women in Metro Vancouver      Principal Investigator:   Yvonne Lamers, PhD   Food, Nutrition & Health  Land and Food Systems - The University of British Columbia    Graduate Student: Jennifer Ho, BSc  Food, Nutrition & Health  Land and Food Systems - The University of British Columbia    Research Assistants: Benny Chan, BSc Food, Nutrition & Health    Land and Food Systems - The University of British Columbia                          153  1.  INTRODUCTION  Vitamin B6 is an essential nutrient important for blood cell formation, a healthy brain, and energy metabolism. Low vitamin B6 status has been associated with an increased risk of several chronic diseases, including cardiovascular disease (also known as heart and vessel disease), atherosclerosis (also known as hardening of the arteries), stroke, colorectal cancer, breast cancer and ovarian cancer. A high prevalence of inadequate dietary vitamin B6 intake was observed in Canadian adult women, which might indicate that this population group is at risk of being vitamin B6 deficient.  To date, little is known about the vitamin B6 status in older adult women in Canada. Thus, it is essential to determine the prevalence of vitamin B6 deficiency in older adult women and to understand the impact of demographic and lifestyle factors on vitamin B6 status. You are being invited to take part in a study that assesses the frequency, as well as potential factors contributing to vitamin B6 deficiency, in older adult women in the Metro Vancouver area.   2. YOUR PARTICIPATION IS VOLUNTARY    Your participation is entirely voluntary, so it is ultimately your decision whether or not to take part in this study. Before you decide, it is important for you to understand what the research involves. This consent form will tell you about the study, why the research is being done, what will happen to you during the study and the possible benefits, risks, and discomforts. If you wish to participate, you will be asked to sign this form. If you decide to take part in this study, you are still free to withdraw at any time. You will not be required to give a reason for your decision to withdraw. If you do not wish to participate, you do not have to provide any reason for your decision not to participate nor will you lose the benefit of any medical care to which you are entitled or are presently receiving. Please take time to read the following information carefully and to discuss it with your family, friends, and doctor before you decide.   At the end of this consent form, there will be the option for giving consent for future use of biological materials (your blood sample) and the option for consenting to being contacted for recruitment in follow up studies. These options are independent of your willingness to participate in the proposed study and it is your decision whether or not you sign the separate consent forms for these options.    3.  WHO IS CONDUCTING THE STUDY?   Researchers from the Food, Nutrition, and Health Department of the Faculty of Land and Food Systems, University of British Columbia, are conducting the study. Funds from The University of British Columbia (new faculty start-up funds) are sponsoring the study.                          154    4.  WHAT IS THE PURPOSE OF THE STUDY?  The goal of this pilot study is to determine the rate of vitamin B6 deficiency in 300 adult women aged 51-70 yr in Metro Vancouver and to determine potential demographic, dietary and lifestyle factors influencing vitamin B6 status in the focus population. This pilot study serves purpose to investigate the dynamics of vitamin B6 status in older adult women. The results gain from this pilot study will serve as a guideline for the design of larger studies in the future.    5. WHO CAN PARTICIPATE IN THIS STUDY?  To be eligible to participate, you must be a healthy woman aged 51-70 yr.   Participants will be exclude if they have chronic conditions (for example: diabetes, cardiovascular disease, cancer, renal failure/kidney disease, chronic obstructive pulmonary disease (COPD), emphysema, bronchitis, arthritis, fibromyalgia, asthma, stroke, thyroid condition, bipolar disorder, schizophrenia, anxiety disorder,  learning disability (attention deficit disorder, attention deficit hyperactivity disorder, dyslexia), eating disorder (anorexia, bulimia), liver disease, gallbladder problems, hepatitis, HIV/AIDS) or gastrointestinal conditions (for example: atrophic gastritis, Cohn’s disease, irritable bowel syndrome, colitis, pernicious anaemia celiac disease, acid indigestion, diverticulitis/diverticulosis, celiac disease, gastric bypass surgery).   Participants who are currently taking any performance enhancing or recreational drugs (for example: steroids, marijuana, cocaine) will be excluded. Participants who are taking any medication that might interact with vitamin B6 (for example: Hydralazine (Apresoline), Phenelizine (Nardil), Tranylcypromine (Parnate), Amiodarone (Cordarone), Phenobarbital (Luminal), Phenytoin (Dilantin), Levodopa, Metformin, anti-Cancer treatment, antibiotics, proton pump inhibitors, antacids, chloramphenicol) will be excluded.    In addition, participants who are unable to provide informed consent, or unable to read and write English are ineligible to participate.    6. WHAT DOES THE STUDY INVOLVE?   The study will be conducted at the Food, Nutrition, & Health Building, University of British Columbia, or at the Clinical Research and Evaluation Unit at the Child and Family Research Institute. The procedures will take about 90 minutes of your time.                          155  Before coming to the study site:  1.!You will be asked to arrive at fasting state, that is, after having fasted for 10 hours. For example, if your visit is scheduled for 8 AM you should not consume anything besides water after 10 PM the night before. You are asked to arrive fasted because eating a meal impacts some of the blood parameters.   2.!For your visit, you are asked to bring in the containers of any supplements (for example vitamin or mineral supplements, multivitamins etc.) that you are currently taking. This includes nutritional supplements such as include nutritional shake (ie. Vega One™), meal replacement drinks (ie. Ensure™), meal replacement bar (ie. Clifbar™), vitamin containing drinks (ie. Vitamin water™). We will send an email prior to the day of the study as a courtesy reminder for this request.  At the study site:  3.!You will be asked to have measured by a female study personnel your height (using a stadiometer), weight (using a scale), waist circumference (using a tape measure) and blood pressure (using a blood pressure moniter). This information will be collected in a private room and will remain confidential.  4.!You will be asked to complete a demographic questionnaire that collects information on age, ethnicity, smoking status, socioeconomic status, and use of hormone therapies. At any time you may refuse to provide any information that you do not feel comfortable sharing.  5.!You will be asked to complete a food frequency questionnaire for assessment of total dietary intake and micronutrient intake.  6.!A tube of fasting blood (less than 24 mL or 5 teaspoons) will be taken from you by a research nurse who is certified to take blood. You will also be asked for a finger prick blood sample. These blood samples will be used to measure blood levels of B-vitamins, related functional biomarkers, and presence of common genetic variants affecting vitamin B6 status. The genetic information gathered in this study will be used for the sole purpose of investigating possible genetic influences of vitamin B6 deficiency. No genetic information will be disclosed to participants and all information will be safeguarded and confidential.   The blood sample will be stored for 25 years at the University of British Columbia (Food Nutrition and Health building, room 250). Dr. Yvonne Lamers will be responsible for the blood sample. The collection of these samples and data will allow us to investigate which factors (including age, ethnicity, or dietary habits) influence someone’s vitamin B6 status and what the prevalence of vitamin B6 deficiency is in Metro Vancouver area. The study protocol has been reviewed and approved by the Clinical Research Ethics Board at the University of British Columbia. The board aims to help protect the rights of research subjects.                        156      7.  WHAT ARE THE POSSIBLE HARMS AND SIDE EFFECTS OF PARTICIPATING?  Taking a blood sample is felt to have very low risks. The needles used to take blood might be uncomfortable and you may feel lightheadedness, and/or get some minor bruising and/or rarely an infection at the site of the blood draw. Some of the questions in the questionnaire ask about personal information (such as ethnicity, income, menstruation and diet history). These questions may be discomforting for some people. Studies involving humans now routinely collect information on race and ethnic origin as well as other characteristics of individuals because these characteristics may influence how people respond to different medications. Providing information on your race or ethnic origin is voluntary.   8. WHAT ARE THE POTENTIAL BENEFITS OF PARTICIPATING?  There may not be direct benefits to you from taking part in this study. We hope that the information learned from this study can be used in the future to help determine the needs for vitamin B6 for older adult women and identify influencing factors that might put individuals at risk of low vitamin B6 status especially for the ethnicities of interest. We hope this study will provide preliminary information that can be used to facilitate further studies in the causes and consequences of low vitamin B6 status.  Upon completion of sample analysis you will receive a detailed document describing your vitamin B6 status as well as information about your dietary vitamin B6 intake. Instructions on how to interpret the results will be included. If you have any questions about the information provided please feel free to contact us. If you have specific nutrition or health related questions upon receiving the results of the study we will recommend that you consult your family doctor and registered dietician. You will be provided with the option of receiving this information in email or paper mail format. Please indicate your preference on the corresponding signature page on Page 9.   9.  REIMBURSEMENT   In order to defray the costs of transport, each participant will receive a $20 gift card. There will be no costs for participants.   10.  WHAT HAPPENS IF I DECIDE TO WITHDRAW MY CONSENT TO PARTICIPATE?                          157  You may withdraw from this study at any time without giving reasons. If you choose to enter the study and then decide to withdraw at a later time, you have the right to request the withdrawal of your information and/or sample collected during the study. This request will be respected to the extent possible. Please note however that there may be exceptions where the data and/or sample will not be able to be withdrawn for example where the data[and/or sample is no longer identifiable (meaning it cannot be linked in any way back to your identity) or where the data has been merged with other data. If you would like to request the withdrawal of your data [and/or samples], please let your study doctor know. If your participation in this study includes enrolling in any optional studies, or long term follow-up, you will be asked whether you wish to withdraw from these as well.    11.  WHAT HAPPENS IF SOMETHING GOES WRONG  By signing this form, you do not give up any of your legal rights and you do not release the study doctor, participating institutions, or anyone else from their legal and professional duties. If you become ill or physically injured as a result of participation in this study, medical treatment will be provided at no additional cost to you. The costs of your medical treatment will be paid by your provincial medical plan.   12. WILL MY TAKING PART IN THIS STUDY BE KEPT CONFIDENTIAL?   Your confidentiality will be respected.  However, research records and health or other source records identifying you may be inspected in the presence of the Principal Investigator or his or her designate and UBC Clinical Research Ethics Board for the purpose of monitoring the research. No information or records that disclose your identity will be published without your consent, nor will any information or records that disclose your identity be removed or released without your consent unless required by law.   You will be assigned a unique study number as a subject in this study. This number will not include any personal information that could identify you (e.g., it will not include your Personal Health Number, SIN, or your initials, etc.). Only this number will be used on any research-related information collected about you during the course of this study, so that your identity [i.e. your name or any other information that could identify you] as a subject in this study will be kept confidential.   Information that contains your identity will remain only with the Principal Investigator and/or designate.  The list that matches your name to the unique study number that is used on your research-related information will not be removed or released without your consent unless required by law.  Your rights to privacy are legally protected by federal and provincial laws that require safeguards to insure that your privacy is respected and also give you the right of access to the information about you that has been provided to the sponsor and, if need be, an opportunity to correct any                        158  errors in this information.  Further details about these laws are available on request to your study doctor.  13.  AFTER THE STUDY IS FINISHED  The collected data will be retained for 25 years. After this period, all information including personal health information will be shredded and destroyed. If you have any questions or desire further information about the study procedures before or during participation, you may contact Dr. Yvonne Lamers, at any time, at 604-827-1776.    14.  WHO DO I CONTACT IF I HAVE QUESTIONS OR CONCERNS ABOUT MY RIGHTS AS A SUBJECT DURING THE STUDY?   If you have any concerns or complaints about your rights as a research participant and/or your experiences while participating in this study, contact the Research Participant Complaint Line in the University of British Columbia Office of Research Ethics by e-mail at RSIL@ors.ubc.ca or by phone at 604-822-8598 (Toll Free: 1-877-822-8598).    15. FOLLOW UP INVESTIGATION  Due to the nature of this study as a pilot investigation there may be opportunity for the researchers to conduct follow up investigations in related areas. Follow up investigation will involve a similar subject group. Your participation in these follow up trials is not required or expected. Your status and environment in this study will not be affected by your willingness to participate in the follow up studies. If you would be willing to consider via signature on page 7. Follow up studies will also explore the general topic of improving our understanding of vitamin B6 deficiency and its impact on health in Canada. All prior confidentiality provisions will still apply.   Please note: There are THREE signature pages to sign. Please consider each signature form individually and only sign if you are comfortable with the conditions that it implies. Remember, you can withdraw your consent to participate in any aspect of the study at any point in the time without explanation.                             159  16. SUBJECT CONSENT TO PARTICIPATE      •! I have read and understood the subject information and consent form.  •! I have had sufficient time to consider the information provided and to ask for advice if necessary.  •! I have had the opportunity to ask questions and have had satisfactory responses to my questions, and I understand that all of the information collected will be kept confidential and that the results will only be used for scientific objectives. •! I understand that my participation in this study is voluntary and that I am completely free to refuse to participate or to withdraw from this study at any time without changing in any way the quality of care that I receive. •! I am not waiving any of my legal rights as a result of signing this consent form, and I understand that there is no guarantee that this study will provide any benefits to me. •! I have read this form and I freely consent to participate in this study. •! I have received a dated and signed copy of this form for my own record.  I      voluntarily give consent to participate in the study.     (Please print your name)  entitled:   Adequacy of Vitamin B6 Status in Older Adult Women  in Metro Vancouver                         Printed name of subject     Signature       Date                          Person obtaining consent, study role                 Signature        Date                             160  17. SUBJECT CONSENT TO BE CONTACTED FOR FOLLOW UP STUDIES   ☐!I  voluntarily give consent to be contacted for recruitment into follow up investigations stemming from this study. The participant will receive a new consent form for any future studies and at that point they can decide whether or not to join the study.   18. SUBJECT CONSENT TO RECEIVE RESULTS   I wish to receive the results by (please check one and provide contact details):  ☐ Email  Please print email address: ________________________________________________________  ☐!Postal mail Please print mailing address: ______________________________________________________                            161  Appendix E  Optional Consent Form for Tissue Banking    Subject Information and Consent Form  OPTIONAL TISSUE BANKING FOR FUTURE RESEARCH  ORIGINAL STUDY TITLE: Adequacy of Vitamin B6 Status in Older Adult Women in Metro Vancouver      Principal Investigator:   Yvonne Lamers, PhD   Food, Nutrition & Health  Land and Food Systems - The University of British Columbia   Graduate Student: Jennifer Ho, BSc  Food, Nutrition & Health  Land and Food Systems - The University of British Columbia    Research Assistants: Benny Chan, BSc Food, Nutrition & Health    Land and Food Systems - The University of British Columbia                          162  1.  INTRODUCTION  In addition to the main part of the research study, you are being invited to participate in optional storage (known as tissue banking) of your blood samples because you are taking part in the main research study titled “Adequacy of Vitamin B-6 Status in Older Adult Women in Metro Vancouver”. You may take part in the main part of the study without having to agree to participate in optional tissue banking.   2.  YOUR PARTICIPATION IS VOLUNTARY  Your participation is entirely voluntary, so it is up to you to decide whether or not to take part in this optional tissue banking. Before you decide, it is important for you to understand what the tissue banking involves. This consent form will tell you about the tissue banking, why it is being done, and the possible benefits and risks to you.   If you wish to participate, you will be asked to sign this form.  If you do decide to take part in this study, you are still free to withdraw at any time and without giving any reasons for your decision. Please take time to read the following information carefully and to discuss it with your family, friends, and doctor before you decide. If you do not wish to participate, you do not have to provide any reason for your decision not to participate nor will you lose the benefit of any medical care, education, or other services to which you are entitled or are presently receiving.   3. WHO IS CONDUCTING THIS STUDY  Researchers from the Food, Nutrition, and Health Department of the Faculty of Land and Food Systems, University of British Columbia, in collaboration with Vancouver Coastal Health are conducting the study. New faculty start-up funds by The University of British Columbia are funding the study. No conflicts of interest to declare.  WHO IS RESPONSIBLE FOR STORING MY BLOOD AND ANY FUTURE RESEARCH USING MY BLOOD? Dr. Yvonne Lamers, PhD, of the Food, Nutrition, and Health Program of the Faculty of Land and Food Systems, University of British Columbia, is responsible for the storage of your blood samples and for any future research involving your samples. Any analysis conducted on these samples will not be the subject of the same Master’s degree project that is associated with the primary study. However, it may be the subject of further graduate projects.   4. BACKGROUND  Left over blood samples taken from you during the primary study would normally be disposed of through the University of British Columbia (UBC) biological waste program.                        163  However, your remaining blood samples can be stored to be used for future research.  This is known as “tissue banking”.  This extra tissue, not needed for a medical diagnosis or treatment, is used by researchers to study nutritional status or disease risk factors and to find better ways to diagnose, prevent, and treat disease in the future.  Dr. Yvonne Lamers, PhD, the researcher of the primary study in which you are participating, is interested in doing research studies in the future on blood samples taken from you that may help to better understand prevention and treatment of B-vitamin related diseases or other nutritional and medical questions not related to B-vitamins. The purpose of such follow-up research would be to address similar overall goals and research areas as this study that were not feasible as part of this project.   5. WHAT IS THE PURPOSE OF THIS STUDY?   The purpose of the tissue banking is to use the blood samples in future research to learn more about the influence of different nutrients on the human body. By understanding how different nutrients levels affect our body, we hope to find new ways to reduce nutrient-related diseases.   6. WHO CAN PARTICIPATE?  Women who are enrolled in the study entitled: “Adequacy of Vitamin B-6 Status in Older Adult Women in Metro Vancouver” and who had blood collected during any study visit can participate in this optional tissue banking part of the primary study.    7. WHO MAY NOT PARTICIPATE IN THE STUDY?  Women who are not enrolled in the study mentioned above may not participate.  8. WHAT IS INVOLVED IN STORING MY BLOOD?   You will not be asked to specifically provide any extra blood samples apart from the 24 mL collected during the main study. Only blood left over after your blood tests in the primary study are completed will be stored.   Your blood samples will be kept under the guardianship of the primary study investigator (Yvonne Lamers), in a secure facility at the Food, Nutrition and Health Building, Room 250, at the University of British Columbia. Technical, physical and administrative safeguards are in place to prevent unauthorized access.  The blood samples will be stored for 25 years, after which they will be discarded through the University of British Columbia biological waste program.   The blood samples will be used for research purposes only and will not be sold. The research done with your samples may or may not help develop commercial products. There are no plans                        164  to provide payment to you if this happens. The blood samples will only be identifiable by assigned subject number and will not be traceable to any personal information.  The principal investigator, responsible graduate student, and research assistants working on the project will have access to the data. Responsibilities concerning privacy and confidentiality will be discussed with the graduate student and research assistants. Any future research studies involving your blood samples will be submitted for review and approval by one of the University of British Columbia’s Clinical Research Ethics Boards.   9. WHAT ARE MY RESPONSIBILITIES?    You have no responsibilities in addition to those involved with the primary study.  10. WHAT ARE THE POSSIBLE HARMS AND SIDE EFFECTS FROM STORING MY BLOOD?   There are no anticipated risks associated with storage of your blood samples.   11. WHAT ARE THE POTENTIAL BENEFITS OF STORING MY BLOOD?   No one knows whether or not you will benefit from this study.  There may or may not be direct benefits to you from taking part in this study.   We hope that the information learned from this study can be used in the future to benefit other people by providing knowledge on adequate nutrient requirements and understanding potential nutrient-disease relationships.    12. WHAT ARE THE ALTERNATIVES TO CONSENTING TO STORAGE OF MY BLOOD SAMPLES?   The alternative is not to consent to the storage of your blood samples. Choosing not to consent will in no way affect the care that you receive or your participation in the primary study.   13. WHAT IF NEW INFORMATION BECOMES AVAILABLE THAT MAY AFFECT MY DECISION TO CONSENT FOR MY BLOOD TO BE STORED?   If new information were to arise that may affect your willingness to consent to storage of your blood samples, this would be made available to you.                         165    14. WHAT HAPPENS IF I DECIDE TO WITHDRAW MY CONSENT FOR STORAGE OF MY BLOOD SAMPLES?   Your consent for the storage of your blood samples is entirely voluntary.  If you decide to consent to the blood storage and later decide to withdraw your consent, you do not have to provide any reason nor will you lose the benefit of any medical care, education, or other services to which you are entitled or are presently receiving. Your participation in the primary study will not be affected. If you wish for your stored blood samples to be destroyed upon your withdrawal, you may contact Dr. Yvonne Lamers, PhD, by phone at 604 827 1776.  The study investigators may choose to discontinue the study at any time or withdraw you from the study at any time if they feel it is in your best interests.  15. WHAT HAPPENS IF SOMETHING GOES WRONG?   Signing this consent form in no way limits your legal rights against the investigators, or anyone else, and you do not release the principal investigator or participating institutions from their legal and professional responsibilities.  16. CAN I BE ASKED TO LEAVE THE STUDY?   If you are not complying with the requirements of the study or for any other reason, the primary investigator may withdraw you from the study and will arrange for your care to continue.  !17. WHAT WILL THE STORING OF MY BLOOD COST ME?   You will not be paid for your blood samples and there will be no cost to you for us to store your blood.     18. WILL MY IDENTITY AND STORAGE OF MY BLOOD SAMPLES BE KEPT CONFIDENTIAL?   Your confidentiality will be respected. However, research records and health or other source records identifying you may be inspected in the presence of the Investigator or his or her designate by representatives of The University of British Columbia Clinical Research Ethics                        166  Board for the purpose of monitoring the research. No information or records that disclose your identity will be published without your consent, nor will any information or records that disclose your identity be removed or released without your consent.    Your blood samples will be stored securely in the Clinical Nutrition Laboratory in the UBC FNH Building and will be identifiable only by a unique study code under the guardianship of Dr. Yvonne Lamers. This code will not include any personal information that could identify you (e.g., it will not include your Personal Health Number, SIN, or your initials, etc.). Only this code will be used on the samples so that your identity [i.e. your name or any other information that could identify you] will be kept confidential.   Information that contains your identity will remain only with the Principal Investigator and/or designate.  The list that matches your name to the unique study code that is used on your blood samples will not be removed or released without your consent.  Your rights to privacy are legally protected by federal and provincial laws that require safeguards to insure that your privacy is respected and also give you the right of access to the information about you that has been provided to the sponsor and, if need be, an opportunity to correct any errors in this information.  Further details about these laws are available on request to your study Principal Investigator, Yvonne Lamers.  It is possible that in the future, other investigators may access your stored blood samples. To ensure your confidentiality your stored blood samples will be identified only by unique ID number, and will have no identifying information attached to it.  19.  WHO DO I CONTACT IF I HAVE QUESTIONS ABOUT STORING MY BLOOD?   If you have any questions or desire further information before or during the time your blood is being stored, you can contact Dr Yvonne Lamers, PhD, at 604-827-1776 or any other members of the study team, listed on the first page of this consent form.   20.  WHO DO I CONTACT IF I HAVE ANY QUESTIONS OR CONCERNS ABOUT MY RIGHTS AS A SUBJECT?   If you have any concerns about your rights as a research subject, contact the Research Participant Complaint Line in the University of British Columbia Office of Research Ethics by e-mail at  RSIL@ors.ubc.ca or by phone at 604-822-8598 (Toll Free: 1-877-822-8598)  21. SUBJECT CONSENT TO PARTICIPATE  OPTIONAL TISSUE BANKING FOR FUTURE RESEARCH                         167   ORIGINAL STUDY TITLE:  ADEQUACY OF VITAMIN B-6 STATUS IN OLDER ADULT WOMEN IN METRO VANCOUVER    22. SIGNATURE  SUBJECT CONSENT TO BLOOD STORAGE    My signature on this consent form means that:  •! I have read and understood this subject information and consent form.  •! I have had sufficient time to consider the information provided and to ask for advice if necessary.  •! I have had the opportunity to ask questions and have received satisfactory responses to my questions. •! I understand that willingness to allow storage and further analysis on my blood samples is voluntary and that I am completely free to refuse this and to withdraw my consent at any time without changing in any way the quality of care I receive.  •! I am not waiving any of my legal rights as a result of signing this consent form,  •! I understand that there is no guarantee that this study will provide any benefits to me.  •! I will receive a signed copy of this consent form for my own records. •! I voluntarily give consent for my blood samples to be stored and analyzed for future research.   I will receive a signed copy of this consent form for my own records.  I consent to participate in this optional blood storage (tissue banking).                   Subject’s Signature   Printed name    Date                           Signature of                  Printed name, Study Role                          Date   Person Obtaining Consent                          168   Appendix F  Study Visit Protocol  Guide to Study Protocol for Participants – UBC Vitamin B6 Status Study Here is a brief guide on what to expect on the day of your visit and some pointers to ensure smooth and timely completion.  1.! The night before your visit please take some time to ensure that your informed consent package is read carefully. Also, ensure that you begin fasting (consuming nothing but water)10 hours before your visit (for example: if your visit is at 9am, please do not consume food or any beverages except water after 11 pm). Please collect all supplements that you consume and bring them with you to the study site. Alternatively, you can take pictures of the supplement, including the product name and label and bring them with you.  Supplements may include multivitamins, isolated vitamin supplements, nutritional shake (ie. Vega One™), meal replacement drinks (ie. Ensure™), meal replacement bar (ie. Clifbar™), vitamin containing drinks (ie. Vitamin water™).  If you are unsure as to whether something qualifies as a ‘supplement’, please contact us or just bring it with you to the study site.  2.! Use the maps provided to plan your transportation to the study site ahead of time. Unfortunately both study centres are within larger campuses and parking may be difficult to locate. If you require assistance planning your travel, please contact us. You will receive a gift certificate to defray the costs of transportation.   3.! Once you have arrived, the study visit will begin!  a.! We will begin by going over the informed consent forms with you. At this point, we will take ample time to clarify any questions you may have. b.! After signing the informed consent, we will begin with a 24 mL (less than 5 teaspoon) blood draw performed by a research nurse or research personnel certified in phlebotomy. c.! We will then ask you to fill out a food frequency questionnaire that asks questions about your nutrient intake. d.! Lastly, we will go over any unfinished sections of the demographic questionnaire with you.   4.! Once all of the questionnaires are completed you will be presented with your gift voucher. At this point, your active participation in this study is completed. The only follow up will be an information package with your vitamin B6 status that will be sent once results are available. If you have any further questions do not hesitate to call research personnel and we will be happy to answer them to the best of our ability.  THANK YOU FOR YOUR PARTICIPATION AND WE LOOK FORWARD TO SEEING YOU AT YOUR STUDY VISIT.                         169   Appendix G  Demographic Questionnaire  *Please note that all information on this questionnaire is labelled with an ID number only, and stored separately from your name and contact details. All information collected will be stored securely, and personally identifiable information will be accessible to no one except Dr Yvonne Lamers.  1| Today’s Date __ __/ __ __ / __ __ __ __(Month/Day/Year)   2| Subject ID Number __ __ __ (###)  3| Location of Subject Visit:   !  Food Nutrition and Health Building, University of British Columbia !  Child and Family Research Institute  4| Height __ __ __ cm / __ __ __ cm (researcher to fill in)   5| Weight __ __ __ kg / __ __ __ kg (researcher to fill in)  6| Waist Circumference __ __ __ cm / __ __ __ cm (researcher to fill in)  7| Hip Circumference __ __ __ cm / __ __ __ cm (researcher to fill in)  8| Blood Pressure __ __ __ / __ __ __ mmHg (researcher to fill in)                               __ __ __ / __ __ __ mmHg (researcher to fill in) ADEQUACY OF VITAMIN B6 STATUS IN OLDER ADULT WOMEN IN METRO VANCOUVER                        170    PERSONAL INFORMATION  9| What is your age? __ __ (##)  ETHNIC BACKGROUND  10| What ethnic group do you identify as:  Please check the MOST appropriate box ! Caucasian/European (01) ! Chinese (02) ! South Asian (03) ! Other (04): ____________________________   11| What area of Metro Vancouver are you from?  Abbotsford, Anmore, Belcarra, Bowen Island, Burnaby, Coquitlam, Delta, Langley – City, Langley – Township, Lions Bay, Maple Ridge, Metro Vancouver, New Westminster, North Vancouver – City, North Vancouver – District, Pitt Meadows, Port Coquitlam, Port Moody, Richmond, Surrey, Vancouver, West Vancouver, White Rock, Electoral Area A ____________________________ (Please fill in 1 from the above list)  SOCIO-DEMOGRAPHIC  12| How many people live in your household?   Please include only individuals (i.e. spouse, partner, family member) who you share income and expenses with. __ __   13| What is your best estimate of the total income, before taxes and deductions, of all household members from all sources in the past 12 months? Only list combined income of individuals (i.e. spouse, partner, other family members) who contribute jointly to the household income and share expenses  ! Less than $15,000 (01)          ! $15,001 to less than or equal to $20,000 (02)                               171  ! $20,001 to less than or equal to $30,000 (03)        ! $30,001 to less than or equal to $40,000 (04)  ! $40,001 to less than or equal to $50,000 (05)  ! $50,001 to less than or equal to $60,000 (06)  ! $60,001 to less than or equal to $70,000 (07)  ! $70,001 to less than or equal to $80,000 (08)  ! $80,000 or more (09) ! Don’t know (Blank)  EDUCATION  14| Please list the highest level of education you have completed to date.  ! Less than secondary (high school) education (01) ! Secondary school (high school) diploma (02) ! Some post-secondary education (03) Trade certificate or diploma, apprenticeship training, or non-university certificate or diploma from community college, CEGEP etc., university education below bachelor’s level (2/3 year diploma or partially completed bachelor’s degree) ! Bachelor’s degree (post-secondary education) (04) ! University degree or certificate higher than bachelor’s degree (05) (i.e. Master, Doctorate, professional degree)  ! Don’t know or decline to respond (Blank)  LIFESTYLE  15| Are you a smoker?   !  Yes, Current frequent smoker (04)    Smoke more than 20 cigarettes per day !  Yes, Current regular smoker (03)  Smoke between 10 and 19 cigarettes per day !  Yes, Current occasional smoker (02)  Smoke between 1 and 9 cigarettes per day !  Former smoker (YES) (01)     !  No, Non-smoker (NO) (00)  FOOD ALLERGIES                        172   16| Are you Lactose Intolerant?  ! Yes, Complete restriction (02)  ! Moderately, Incomplete restriction (01)       ! No, No (Zero) restriction (00)  MENSTRUAL CYCLE  17| Are you still menstruating?  !Yes (00)                 !No (01)  JUMP TO QUESTION 22  MENSTRUATING  18| If you answered YES to the last question - What was the approximate date of your last period (the last date that you menstruated)?  Month/Day __ __/__ __  19| Do you use hormonal contraceptives?  i.e. birth control pill, birth control patch, depo provera etc.?  !Current user (Yes) (02)                 !Former user (Yes) (01) !Never (No) (00)  20| If you answered YES to the last question - Approximately how long have you been taking hormone contraceptives?  __ __(years)/__ __ (months)  21| If you answered YES to question 19 – How often do you take hormone contraceptives?  …………………………………………                         173  MENOPAUSE  22| If you answered NO to question 17 - What was the approximate date of your last period (the last date that you menstruated)?  Month/Day/Year __ __/__ __/__ __  23| Did you use hormonal contraceptives when you were menstruating?  i.e. birth control pill, birth control patch, depo provera etc.?  ! Yes (01)                 ! No (00)  24| Do you use menopausal hormonal therapy?  i.e. hormone therapy (HT), estrogen therapy (ET) etc.?  ! Current user (Yes) (02)                 ! Former user (Yes) (01) ! Never (No) (00)  25| If you answered YES to the last question - Approximately how long have you been taking menopausal hormone therapy?  __ __(years)/__ __ (months)  26| If you answered YES to question 24 – How often do you take hormone medicine?  …………………………………………….  SUPPLEMENT USE  27| Do you use nutritional vitamin or mineral supplements?  - Vitamin supplement i.e. vitamin C, D, B6, B12 or folate supplements  !  Yes (01) !  No (00)                         174  -! Mineral supplement i.e. ferritin, calcium, zinc supplement  !  Yes (01) !  No (00)  -! Multivitamin supplement i.e. centrum, calcium plus vitamin D  !  Yes (01) !  No (00)  -! Other food Supplement i.e. glucosamine, probiotics, shakes  !  Yes (01) !  No (00)  28| Do you use supplements containing Vitamin B6 (Pyridoxine)?  !  Yes (01) !  No (00)  29| Do you use supplements containing Folate (Folic Acid, Vitamin B9)?  !  Yes (01) !  No (00)  30| Do you use supplements containing Vitamin B12 (Cobalamin)?  !  Yes (01) !  No (00)   PLEASE COMPLETE ONE SECTION FOR EACH SUPPLEMENT YOU CONSUME  1.! Type of supplement:  !  Vitamin supplement i.e. vitamin C, D, B6, B12 or folate supplements !  Mineral supplement i.e. ferritin, calcium, zinc supplement !  Multivitamin supplement i.e. centrum, calcium plus vitamin D !  Other food Supplement i.e. glucosamine, probiotics, shakes  Product name  ……………………………………….                        175    How often do you consume it? (i.e. Daily, weekly, x times per week)  …………………………………………  Number of doses every time (i.e. Tablet, scoop, bottle, bar)  …………………………………………  Dose per serving  ………………………………..Vitamin B-6 (in micrograms)  ………………………………..Folate (in micrograms)  ………………………………..Vitamin B-12 (in micrograms)    2.! Type of supplement:  !  Vitamin supplement i.e. vitamin C, D, B6, B12 or folate supplements !  Mineral supplement i.e. ferritin, calcium, zinc supplement !  Multivitamin supplement i.e. centrum, calcium plus vitamin D !  Other food Supplement i.e. glucosamine, probiotics, shakes  Product name  ……………………………………….   How often do you consume it? (i.e. Daily, weekly, x times per week)  …………………………………………  Number of doses every time (i.e. Tablet, scoop, bottle, bar)  …………………………………………  Dose per serving  ………………………………..Vitamin B-6 (in micrograms)  ………………………………..Folate (in micrograms)                         176  ………………………………..Vitamin B-12 (in micrograms)   3.! Type of supplement:  !  Vitamin supplement i.e. vitamin C, D, B6, B12 or folate supplements !  Mineral supplement i.e. ferritin, calcium, zinc supplement !  Multivitamin supplement i.e. centrum, calcium plus vitamin D !  Other food Supplement i.e. glucosamine, probiotics, shakes  Product name  ……………………………………….   How often do you consume it? (i.e. Daily, weekly, x times per week)  …………………………………………  Number of doses every time (i.e. Tablet, scoop, bottle, bar)  …………………………………………  Dose per serving  ………………………………..Vitamin B-6 (in micrograms)  ………………………………..Folate (in micrograms)  ………………………………..Vitamin B-12 (in micrograms)   4.! Type of supplement:  !  Vitamin supplement i.e. vitamin C, D, B6, B12 or folate supplements !  Mineral supplement i.e. ferritin, calcium, zinc supplement !  Multivitamin supplement i.e. centrum, calcium plus vitamin D !  Other food Supplement i.e. glucosamine, probiotics, shakes  Product name  ……………………………………….   How often do you consume it? (i.e. Daily, weekly, x times per week)                         177  …………………………………………  Number of doses every time (i.e. Tablet, scoop, bottle, bar)  …………………………………………  Dose per serving  ………………………………..Vitamin B-6 (in micrograms)  ………………………………..Folate (in micrograms)  ………………………………..Vitamin B-12 (in micrograms)   End of questionnaire – Thank you for your participation.                          178  Appendix H  Result Letter Dear (name of person),  Thank you for participating in the UBC Vitamin B6 Status Study.  It is with pleasure that we wish to share the results of your blood sample with you. Please find underneath the results for your blood test, along with some general information about the blood indicators.  If you have any questions, comments, or concerns, after reading this message, please do not hesitate to contact us. Thank you again for your participation.  1. The result from your vitamin B6 screening indicated that  o! Your vitamin B6 status is adequate.   o! Your vitamin B6 status is suboptimal. Please consult your family doctor or dietitian.   o! You are deficient in vitamin B6. Please consult your family doctor or dietitian as you may be at risk for some health problems such as neurological decline or anemia. !Vitamin B6 plays an important role in maintaining good health by contributing to a healthy brain, metabolism, and energy levels. The body uses vitamin B6 to make and use protein and glycogen, which is the stored energy in your muscles and liver. Vitamin B6 also helps form hemoglobin, which carries oxygen in your blood. Severe deficiency may lead to anemia, seizures, convulsion, and peripheral neuropathy.  2. The assessment of your dietary vitamin B6 intake showed the following:   Your average daily intake of vitamin B6 is ……………………….milligrams. Thus, based on the output of your diet history questionnaire, the results suggest that                         179  o! you are likely not getting enough vitamin B6 from your diet. Please seek out the advice of a family doctor or registered dietitian.  o! you are likely meeting the dietary requirements for vitamin B6.   It is recommended that women aged above 50 years get 1.5 mg of vitamin B6 daily. Foods with vitamin B6 are abundant and the good food sources with high levels of vitamin B6 are meat, fish, poultry, organ meat, fortified cereals, soy products, nuts, and some vegetables and fruits.   3. The result from your vitamin B12 screening indicated that  o! Your vitamin B12 status is adequate.   o! Your vitamin B12 status is suboptimal. Please consult your family doctor or dietician.   o! You are deficient in vitamin B12. Please consult your family doctor or dietician as you may be at risk for some health problems such as anemia or neurological decline.  Vitamin B12 plays a vital role in maintaining good health by contributing to a healthy brain, metabolism, and energy levels. It is protective against neurological decline, pregnancy complications, and anemia. It is recommended that older adult women get 2.4 µg of vitamin B12 daily. Foods with vitamin B12 are limited and the most potent supply is from animal sourced foods (meat, cheese, milk, eggs, seafood). If you do not consume animal foods fortified products may be a positive alternative as plants do not contain B12.  4. The assessment of your dietary vitamin B12 intake showed the following:   Your average daily intake of vitamin B12 is ……………………….micrograms. Thus, based on the output of your diet history questionnaire, the results suggest that                         180  o! you are likely not getting enough vitamin B12 from your diet. Please seek out the advice of a family doctor or registered dietitian.  o! you are likely meeting the dietary requirements for vitamin B12.   The daily recommendations for vitamin B12 intake for women of your age is 2.4 micrograms per day. Dietary sources of B12 include but are not limited to meat, fish, milk and other dairy products, eggs, and fortified foods. Please keep in mind that the diet history questionnaire used to determine your dietary vitamin B6 and B12 intake provides a ballpark number, or estimation of your typical dietary habits. Your actual intake may vary slightly and may be different on a day-to-day basis. Thank you again for your participation,  Jennifer Ho, BSc – Graduate Student   Dr. Yvonne Lamers, PhD – Principal Investigator    

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