Cork Centre for Vitamin D and Nutrition Research - Doctoral Theses

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    Quantifying minor vitamin D metabolites using liquid chromatography-tandem mass spectrometry and determining their contribution to vitamin D nutritional status
    (University College Cork, 2019) Dowling, Kirsten; Cashman, Kevin; Kiely, Mairead; European Commission; Seventh Framework Programme; Department of Agriculture, Food and the Marine; Nordisk Ministerråd
    Adequate vitamin D status is necessary throughout life in terms of maintaining bone strength, calcium and phosphorus balance, as well as prevention of rickets in children and osteomalacia in adults. The accepted biomarker used for the assessment of vitamin D status is circulating total 25-hydroxyvitamin D (25(OH)D) concentration, which is comprised of the sum of 25-hydroxyvitamin D3 (25(OH)D3) and 25-hydroxvitamin D2 (25(OH)D2) The use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of total 25(OH)D has also enabled the detection of low concentration, minor vitamin D metabolites in serum. The aims of this work were to examine the impact of three such minor vitamin D metabolites, 25(OH)D2, 24,25-dihydroxyvitamin D (24,25(OH)2D), and 3 epimer of 25(OH)D3 (3-epi-25(OH)D3) on the assessment of vitamin D nutritional status and also to provide further insight and refinement of our understanding of the vitamin D metabolic pathway. This required the development and validation of LC-MS/MS methods for measuring these three metabolites in multiple matrices (serum/plasma, meat, milk). Serum 25(OH)D2, not only contributes to the total 25(OH)D estimate, but also serves as a marker for vitamin D2 intake. Its presence in sera from a diverse collection of European populations, as part of this work, suggests that vitamin D2 is present in the food chain. Preliminary analysis of Irish milk and beef, as prioritized candidate food sources, for 25(OH)D2 and vitamin D2 were unable to pinpoint the primary dietary contributors to vitamin D2 intake. In the present work, an improved method for LC-MS/MS analysis was developed which produced accurate results for both total 25(OH)D and the metabolite 24,25(OH)2D. Application of this method allowed us to demonstrate that the presence of 24,25(OH)2D in serum led to an overestimated measurement of serum total 25(OH)D when employing an exemplar immunoassay technique. 24,25(OH)2D is the first metabolite in the vitamin D catabolic cascade, and thus considered a marker of vitamin D catabolism. The new LC-MS/MS method for 24,25(OH)2D was applied as a means to determine differential catabolic rates between 25(OH)D2 and 25(OH)D3 in multiple species. This analysis also highlighted novel inter-species differences in the ratio of 24,25(OH)2D to 25(OH)D. The availability of a large sample set of age-stratified population studies across Europe revealed that serum 3-epi-25(OH)D3 was primarily dependent on age, with concentrations highest at birth and decreasing steadily up until adolescence. While some 3-epi-25(OH)D3 was detected in pork and beef, as potential exogenous sources of the epimer, the majority of circulating 3-epi-25(OH)D3 is produced endogenously via an enzyme distinct from 25-hydroxlase. Overall, this work highlights the potential of using LC-MS/MS to analyse minor vitamin D metabolites which can help add new information to the research area of vitamin D metabolism and status, and potentially better inform clinical interpretations of vitamin D status.
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    An exploration of the calcium metabolic system in pregnancy and public health programmes to improve early nutrition in maternal-infant cohorts
    (University College Cork, 2019) Hemmingway, Andrea; Kiely, Mairead; Science Foundation Ireland
    Although pregnancy and infancy represent periods of particular nutritional vulnerability, many gaps in the evidence base remain during these life-stages. The research in this thesis was conducted in two phases across three studies in pregnant women and infants. The aim of the first phase was to examine the vitamin D-calcium metabolic system in pregnancy. In the largest clinical study to date, serum 25- hydroxyvitamin D [25(OH)D] and parathyroid hormone [PTH] were analysed in 1754 women in the SCOPE pregnancy cohort. While 25% of participants had a 25(OH)D concentration > 75 nmol/L, 17% were < 30 nmol/L. Functional vitamin D deficiency, defined as 25(OH)D < 30 nmol/L plus elevated PTH (> 80th percentile), occurred in 5.5% of participants. The prevalence of elevated mean arterial pressure (19.1 vs. 9.7%) and small-for-gestational-age (16.0 vs. 6.7%) were highest in those with functional vitamin D deficiency (P < 0.05), compared with the reference group [25(OH)D ≥ 75 nmol/L and normal PTH]. The adjusted prevalence ratio and risk ratio (95% CIs) were 1.83 (1.02, 3.27) and 1.53 (0.80, 2.93) for elevated mean arterial pressure and small-for-gestational-age, respectively. These data indicate the need to consider the broader calcium metabolic system in future studies of vitamin D and perinatal health. Following this, the relative impact of serum 25(OH)D and calcium intake on PTH was explored using baseline data from a vitamin D dose-response trial (n = 142). PTH was inversely correlated with 25(OH)D (r = -0.311, P < 0.001), but not calcium intake (r = -0.087, P = 0.306). In categorical analyses, there was no 25(OH)D-calcium interaction effect on PTH (P = 0.941) and 25(OH)D < 50 nmol/L, but not stratified calcium intake, significantly affected PTH concentration (P = 0.025 and 0.822, respectively). In this group of white-skinned pregnant women, who generally had sufficient calcium intakes, serum 25(OH)D was important for maintaining PTH concentration. The background nutritional status and participant population appear to be critical factors. The second research phase explored early life nutrition in the COMBINE birth cohort. COMBINE, a prospective, nutrition-led study, recruited 456 participants between 2015 and 2017 and has 9 study visits over the first 2 years of life. Examination of infant feeding practices indicated that 44% gave breastmilk as the main milk source at hospital discharge, 36% at 2 months, 24% at 6 months and 19% at 9 months. The rate of infant formula only feeding increased from 25% at discharge to 49% at 2 months and 74% at 9 months of age. Combination feeding of breastmilk and infant formula was common at discharge (31%) and 1 month (20%); these mothers were more likely to stop breastfeeding altogether than those who breastfed (all P< 0.05). The median (IQR) age of introduction to solid foods was 21 (18, 24) weeks; 9% introduced solids before 17 weeks of age. While these data provide evidence of some progress towards longer breastfeeding durations, there remains much scope to improve infant feeding practices in Ireland. Adherence to the national infant vitamin D supplementation policy, implemented in 2010, is not monitored nationally and the policy has not been formally evaluated; policy adherence was examined in the COMBINE cohort (n = 365). Most parents initiated supplementation at birth (92%), used a vitamin D3 only product (94%), and gave the recommended 5 µg dose (88%). Half (51%) administered vitamin D daily and a further 33% supplemented at least 3-6 times/week. Overall, 30% adhered fully to the policy, providing 5 µg vitamin D3 daily from birth to 12 months. Given high levels of maternal and infant deficiency and the current lack of a maternal supplementation policy in Ireland, the infant vitamin D supplementation policy should remain a public health priority. With two research foci, the data generated in this thesis provides much-needed advancement of knowledge regarding the vitamin D-calcium metabolic system in pregnancy and public health nutrition in the first year of life.