Yvonne Lamers

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Background: Early detection of vitamin B-12 (B-12) deficiency through reliable biomarkers is critical to prevent poor development and life-long health consequences. Children with short bowel syndrome (SBS) are at risk of B-12 deficiency. Serum total B-12 is the most commonly used B-12 biomarker but there is ambiguity to its performance. Holotranscobalamin (holoTC), the B-12 form taken up by cells, may be a more sensitive biomarker. Methylmalonic acid (MMA) is a functional and sensitive biomarker, but lacks availability in clinical laboratories. Overall, the utility of B-12 biomarkers in the pediatric population is limited, in part due to the lack of age-specific cutoffs. Objectives: 1) To derive age- and sex-specific reference intervals for serum holoTC and MMA concentrations in healthy children, and 2) to compare the diagnostic ability of total B-12 and holoTC to detect functional B-12 deficiency in pediatric SBS patients.Methods: The project consisted of 1) the secondary analysis of bio-banked serum samples for MMA and holoTC from 337 healthy Canadian children aged 0-18 years, and of 2) a descriptive, prospective study with 26 SBS patients from the BC Children’s Hospital in Vancouver, BC. Clinical and dietary data, and blood samples for quantitation of B-12 biomarkers, were collected over 2 years. Results: 1) Age-group partitions but no sex partitions were identified for MMA and holoTC. Upper reference limits (97.5th percentiles) for MMA were calculated for infants aged 0-1 year and children aged 1-19 years, and lower reference limits for holoTC were estimated for infants aged 0-1 year, children aged 1-14 years and adolescents aged 14-19 years. 2) The diagnostic ability to detect functional B-12 deficiency was higher for holoTC compared to total B-12. HoloTC identified 4 out of 5 cases and total B-12 identified 1 of 5 cases of elevated MMA in our cohort of children with SBS. Conclusion: This is the first study to provide age-specific reference intervals for serum MMA and holoTC, that are required given the high variability of MMA and holoTC concentration across childhood. Serum holoTC may be a more reliable indicator than serum total B-12 at identifying functional B-12 deficiency in children with SBS.

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Vitamin B12 (B12) is an essential nutrient required for optimal energy metabolism and nervous system functioning. Adults aged >50 years are at risk of impaired B12 digestion and absorption and are recommended to consume B12-fortified foods and/or supplements, i.e. sources of free B12. The general consensus is that B12 has high bioavailability from dairy sources; therefore, developing a fortified dairy product represents a promising strategy to fill a market niche and improve B12 status in older adults.This research thesis consists of three main phases, with the following objectives: (I) to identify the most suitable method for the analysis of added B12 in yoghurts; (II) to develop B12-fortified yoghurts and assess shelf-life stability; and (III) to assess the efficacy of consuming one daily portion of B12-fortified, versus unfortified yoghurt on the B12 status of healthy older adults.For objective I, I developed a High-Performance Liquid Chromatography method and compared its performance to that of RIDASCREEN®, an immunoassay. I found that RIDASCREEN® was the most suitable method for measuring B12 in yoghurt, given its lowest level of quantitation (0.5 μg/L).For objective II, the shelf-life stability of methylcobalamin (MeCB), a naturally-occurring B12 form, and cyanocobalamin (CnCB), the synthetic B12 form, added to yoghurts either in isolated or encapsulated form was tested by measuring total B12 concentrations in yoghurts for 8 weeks. Results indicated that CnCB was a stable fortificant throughout yoghurt shelf-life, and isolated MeCB was stable up until week 4.For objective III, I performed an 8-week double-blind, randomized controlled intervention trial. Yoghurts were fortified with 50 μg MeCB. B12 status was measured at baseline, weeks 4 and 8 using serum total B12 concentration. In a total of 66 participants (aged 50-74y), a significant time-treatment interaction was found (p0.001). After 4 weeks, serum total B12 concentration increased by 47% in the fortified group, and 8% in the control group, with no changes between weeks 4 and 8. This study brings novel findings to applied food science and human nutrition, by showing the feasibility of developing a B12-fortified yoghurt, which was efficacious in increasing serum total B12 concentration of older adults.

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Background: Riboflavin, or vitamin B-2, functions as a coenzyme in numerous metabolic pathways and is essential for adequate cell growth. Riboflavin status has been shown to be inversely associated with the risk of cardiovascular diseases and cancers. Erythrocyte glutathione reductase activation coefficient (EGRac) is a functional indicator and considered as the ‘gold standard’ biomarker of riboflavin status. Previous studies have reported a high prevalence of biochemical riboflavin deficiency but a low prevalence of dietary inadequacy in adults aged ≥65 years. In Canada, 3% of adult women aged 51-70 years had inadequate dietary riboflavin intake; however, data are lacking on biochemical riboflavin status.Objectives: The objectives of this research were to determine the biochemical riboflavin status, and to identify the dietary, demographic and lifestyle predictors of riboflavin status in adult women (aged 51-70 years) in Metro Vancouver.Methods: This secondary analysis used data and biospecimens from a cross-sectional study of a convenience sample of 223 adult women age 51-70 years. A fasting blood sample, blood pressure measurements, sociodemographic, anthropometric and dietary intake data were collected during a single-day study visit. Riboflavin status was measured using EGRac. Plasma riboflavin concentration was analyzed using an in-house validated liquid chromatography-tandem mass spectrometry assay.Results: Overall, 29% of the study population had riboflavin deficiency (EGRac ≥1.4) and 22% had suboptimal status (EGRac ≥1.3 and 1.4). However, only 4.7% did not meet their dietary riboflavin requirements (i.e. riboflavin intake ≥0.9 mg/day). Riboflavin intake from food and supplements was significantly correlated with EGRac (Spearman-rho=-0.55; p0.001), and plasma riboflavin concentration (Spearman-rho=0.45; p 0.001). Total riboflavin intake and Chinese ethnicity were significant predictors of biochemical riboflavin status. Conclusion: This study is the first to determine biochemical riboflavin status of free-living adult women aged 51-70 years in Metro Vancouver and Canada. It provides new information and preliminary data for future research including information on possible predictors of riboflavin deficiency in this population group. High prevalence of biochemical riboflavin deficiency and suboptimal status among the study population may be a cause for concern. The discrepancy between the diagnosis of biochemical riboflavin deficiency and total riboflavin intake inadequacy warrants more research.

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Vitamin B₁₂ (B₁₂), a coenzyme required for DNA synthesis, methylation, and myelination, is important for fetal growth and development. Lower maternal B₁₂ status has been associated with preterm birth (37 gestational weeks) and low birth weight (2,500 grams), which are linked to morbidity and mortality across the lifespan. In Canada, 17-25% of women in early pregnancy were classified as B₁₂ deficient (serum total B₁₂ concentration 148 pmol/L) and maternal total B₁₂ concentration decreases throughout pregnancy; however, there is limited research on whether maternal B₁₂ status is associated with birth outcomes in Canadian pregnant women. This study aimed to determine the association between maternal B₁₂ status and birth outcomes and newborn functional B₁₂ status, and to identify predictors of newborn functional B₁₂ status. A secondary analysis of 709 mother-newborn pairs in British Columbia (BC), Canada, was conducted. Bio-banked first- (n=656) and second-trimester (n=709) maternal serum samples of apparently healthy South Asian (50%) and European (50%) women from the BC Prenatal Genetic Screening Program were quantified for B₁₂ biomarkers (total B₁₂, holotranscobalamin (holoTC), methylmalonic acid (MMA) and total homocysteine (tHcy)). Functional newborn B₁₂ status was indicated by MMA quantified in dried blood spots (DBSs). Obstetric history and birth outcome data were obtained from the BC Perinatal Data Registry. All associations were determined using multiple linear regression. Maternal B₁₂ biomarker concentrations were not linearly associated with birth weight z-score, head circumference z-score and gestational age at birth. However, a 10-pmol/L increase in first- and second-trimester maternal total B₁₂ and holoTC were associated with a 0.5-0.6% and 2.4-2.8% decrease in newborn DBS MMA, respectively; and a 10-nmol/L increase in first- and second-trimester maternal MMA and a 1-μmol/L increase in second-trimester tHcy with a 0.7-0.8% and 3% increase in newborn DBS MMA, respectively. Predictors of newborn DBS MMA concentration include maternal MMA concentration in both trimesters, first-trimester holoTC and second-trimester total B₁₂, hypertensive disorder of pregnancy, parity, newborn sex, newborn feeding following delivery, gestational age at birth and neonatal age at DBS collection. Further research in women at high risk of adverse birth outcomes, and the association between newborn MMA and functional outcomes is needed.

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Birthweight and gestational age at birth have been inversely associated with chronicdisease risk in later life. The vitamins folate and vitamin B12 (B12) have interdependentmetabolic functions that are essential for fetal growth. Maternal folate and B12 concentrationsduring pregnancy have been positively associated with birthweight and gestational age at birth;however, the findings from the literature are inconsistent. The objective of this research was toevaluate the association of maternal serum folate and B12 biomarker concentrations, combinedand individually, with birthweight and gestational age at birth.This retrospective cohort study included biobanked non-fasting serum samples and datafrom 674 apparently healthy pregnant women of South Asian and European ethnicity residing inLower Mainland, British Columbia (BC). Maternal serum samples, collected in the first andsecond trimesters of pregnancy, were retrieved from the BC Prenatal Genetic Screening Programand analysed for folate and B12 biomarker concentrations. Birth outcome data were retrievedfrom the BC Newborn Screening Program. The association of folate and B12 biomarkerconcentrations with birth outcomes was assessed using multiple linear regression models withadjustment for confounding factors, including infant sex, ethnicity and maternal age.The prevalence of low birthweight, preterm and small-for-gestational-age were 1.9%,8.9% and 0.88%, respectively. The combined maternal folate and B12 status, in either trimester,was not associated with birthweight or gestational age at birth. Maternal B12 biomarkerconcentrations individually, in either trimester, were not or only weakly associated with birthoutcomes. Second-trimester maternal folate concentrations of the second, third and fourthquartile group (Q2=55.5-69.3, Q3=69.3-87.8, Q4≥87.8 nmol/L, respectively) were associatedwith an approximate 0.6-week (i.e., 4-day) increase in gestational age at birth, compared to theiiireference group (Q1≤55.5 nmol/L) (95% CI: 0.28, 1.02, p=0.02; 95% CI: 0.23, 0.95, p=0.03,95% CI: 0.16, 0.89, p=0.005, respectively).In conclusion, early pregnancy folate and B12 status were neither combined norindividually associated with birthweight in this sample. Due to the vitamins’ importance in fetalgrowth and development, the association between maternal folate and B12 status and birthoutcomes warrants further investigation in a population with a higher prevalence of lowbirthweight and preterm birth.

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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 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.

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Low maternal vitamin B12 (B12) status has been associated with an increased risk for adverse offspring health outcomes, including neural tube defects and insulin resistance. High rates of B12 deficiency have been reported in South Asians, who comprise one of Canada’s largest minority groups, and Canadian women of childbearing age. Comprehensive B12 status assessment should include multiple biomarkers to reduce the risk of misclassification. However, there is no consensus on the appropriate cut-off values to define chronic and marginal B12 deficiency. Our goal was to assess the rate and determinants of B12 deficiency in healthy South Asian and European women in Metro Vancouver using multiple biomarkers. We conducted a cross-sectional descriptive study in a convenience sample (n=207) of South Asian and European women (19-35y). Anthropometric measurements and questionnaire data on demographics, lifestyle and diet were collected. Vitamin B12 status was assessed using serum vitamin B12 (SB12), serum holotranscobalamin (holoTC) and plasma methylmalonic acid (MMA) as biomarkers. The association of lifestyle, social, dietary, and genetic variables with B12 status was examined using multiple regression models. Using conventional SB12 concentration cut-offs, 14% of participants with biochemical data (n=204) were classified with chronic deficiency (SB12 148 pmol/L), and 20% with marginal deficiency (SB12 148-220 pmol/L). There were no ethnic differences in rates of deficiency. The rate of B12 deficiency dropped substantially (5-12% decrease) when cut-offs for alternate biomarkers (holoTC and MMA) were applied alone or in combination with SB12 to indicate functional deficiency. Vitamin B12 status was influenced positively by dietary B12 intake and B12 supplement use, and negatively by oral contraceptive use and first generation immigrant status. We observed a high rate (34%) of B12 inadequacy based on SB12 levels, indicating a potential need for peri-conceptional monitoring of B12 status. Risk factors associated with B12 deficiency should be further assessed to determine whether surveillance of at-risk groups is required. Multiple biomarkers should be applied henceforth in B12 status assessment, as SB12 alone may lack accuracy and may not be sensitive for detecting marginal deficiency. As such, further investigation into the appropriate cut-offs for MMA and holoTC is needed.

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