Essential trace element analysis of human breast milk. Nor Hidayah Mohd Taufek B.Pharm (Hons), MPH. A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 School of Pharmacy Abstract Introduction: Recent estimates in 2010 for the number of infants born prematurely, defined as those born before 37 weeks gestation, was 15 million worldwide. The complications of preterm birth can be extensive and include low birth weight, respiratory complications, cardiac defects, necrotising enterocolitis, sepsis, and intraventricular haemorrhage. Preterm infants have lower body reserves of many nutrients compared to term infants, that can lead to poor weight gain, growth and development. Human milk is recognised as the optimum source of nutrition to ensure normal growth and development in infants. It contains a unique composition of macronutrients (fat, protein), micronutrients (vitamins, minerals) as well as immunological components. Women who deliver prematurely often cannot produce sufficient quantities of milk for their infant initially. This has led to the development of milk banks worldwide, where milk can be donated by women meeting certain criteria. In most cases the donated milk is pasteurised and frozen until required. Such milk is known as pasteurised donor human milk (PDHM) and is considered to be life saving for many premature infants. Essential trace elements are vital in early neonatal nutrition to promote normal growth. Trace elements are present in human milk but information regarding optimal concentrations in milk is lacking. Trace element deficiencies although rare, have been reported in exclusively breastfed infants and can lead to adverse health outcome if left untreated. The composition of preterm milk is recognised to be different to that of term milk. Studies are lacking investigating trace element concentrations in preterm milk compared to term milk, and if concentrations of trace elements vary over lactation stage, as is the case with other components of milk such as protein and fat. ii Aim: To investigate essential trace element content of human breast milk. Research objectives: 1. To validate a robust, reproducible and sensitive method to simultaneously determine the concentration of trace elements in human milk. 2. To investigate the effect of the pasteurisation process on the concentration of trace elements in donor human milk. 3. To explore the effect of lactation stage on the concentration of trace elements in human milk. 4. To investigate differences in the concentration of trace elements between preterm milk and term milk. Methods: The studies undertaken for this thesis received ethical approval from the Royal Brisbane & Women’s Hospital (RBWH) and the University of Queensland Ethics Committee. A fully validated, robust and reproducible method was established to simultaneously analyse and quantify eight essential trace elements in small samples of human milk, using inductively coupled plasma mass-spectrometry. To investigate the effects of pasteurisation in human milk, 16 women were recruited to the study and analysis of pre- and post-pasteurisation samples was undertaken. To investigate any variation in concentration of trace elements in human milk over time, a case study of one mother who delivered an extremely preterm neonate was included looking at samples taken over 10 weeks postpartum. To investigate any differences between preterm milk and term milk, women (21 preterm and 9 term) donated expressed breast milk (EBM) over the first three months postpartum. Results: The validated method was able to quantify eight essential trace elements in 0.2 mL samples of human milk: zinc, copper, selenium, manganese, iodine, iron, iii molybdenum and bromine. This method was the first to report bromine, a newly recognised essential element in human milk. The Holder pasteurisation process used to reduce the bacterial load of donor milk had minimal effects on the concentration of trace elements, except for iron (p < 0.05). The case study of a mother delivering an extremely premature neonate (24 weeks gestation) revealed that certain trace elements may be present in breast milk at suboptimal concentrations for normal health and development in the neonate. During the first three months postpartum, preterm milk samples (n = 384) and term milk samples (n = 165) were compared. Higher median zinc concentrations were observed in preterm (3647 µg/L) compared to term samples (2478 µg/L) in the first month postpartum. Similar concentrations in both groups were observed for selenium, manganese, iodine, iron, molybdenum and bromine during the first three months postpartum. A general decrease in concentration was observed for zinc, copper and bromine over the same period. Conclusion: The newly validated analytical method using alkaline dissolution was suitable for use in clinical application investigating trace element concentrations in small volume human milk samples. The Holder pasteurisation process used in the preservation of donor human milk did not cause significant loss of trace elements, except for iron. Few differences were found in the concentration of trace elements between preterm and term milk overall, except for zinc, but further work is needed to establish these findings. Preterm and term milk exhibited a general decline in zinc, copper and bromine concentrations over lactation stage but relatively stable concentrations were found for selenium, manganese, iodine, iron and molybdenum. iv Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. v Publications during candidature The following is a list of publications during candidature: Conference abstracts: N. Mohd Taufek, D. Cartwright, M. Davies, A.K. Hewavitharana, P. Koorts, H. McConachy, P. N. Shaw, K. Whitfield. Investigating the effect of the pasteurisation process on trace elements in donor breast milk, at Conference on Neonatology, 25th-27th June 2015 in Avignon, Provence, France. N. Mohd Taufek, D. Cartwright, M. Davies, A.K. Hewavitharana, P. Koorts, H. McConachy, P. N. Shaw, K. Whitfield. Investigating the effect of the pasteurisation process on trace elements in donor breast milk, at Neonatal & Paediatric Pharmacist Group conference, 6th-9th November 2015, United Kingdom. N. Mohd Taufek, D. Cartwright, M. Davies, A.K. Hewavitharana, P. Koorts, K. Palmer-Field, P. N. Shaw, K. Whitfield. Investigating the variation of eight essential trace elements in preterm breast milk in the first month of lactation, at The Neonate – An International Symposium for Asia, 30th March - 1st April 2016, Shanghai, China. N. Mohd Taufek, J. A. Duley, H. G. Liley, P. N. Shaw, K. Whitfield. The longitudinal variation of eight essential trace elements in preterm human milk – a case study, at The Neonate – An International Symposium for Asia, 30th March - 1st April 2016, Shanghai, China. vi Peer-reviewed papers: Nor Mohd-Taufek, David Cartwright, Mark Davies, Amitha K. Hewavitharana, Pieter Koorts, Paul N. Shaw, Ronald Sumner, Eugene Lee, Karen Whitfield (2016). The simultaneous analysis of eight essential trace elements in human milk by ICP-MS, Food Analytical Methods, 2016:9(7):2068-2075.doi: 10.1007/s12161-015-0396-z. Nor Mohd-Taufek, David Cartwright, Mark Davies, Amitha K Hewavitharana, Helen McConachy, Pieter Koorts, Paul N Shaw, Ronald Sumner, Karen Whitfield (2016). The effect of pasteurization on trace elements in donor breast milk, Journal of Perinatology, 2016:36:897–900. doi:10.1038/jp.2016.88. vii Publications included in this thesis Nor Mohd-Taufek, David Cartwright, Mark Davies, Amitha K. Hewavitharana, Pieter Koorts, Paul N. Shaw, Ronald Sumner, Eugene Lee, Karen Whitfield. The simultaneous analysis of eight essential trace elements in human milk by ICP-MS, Journal of Food Analytical Methods, 2016:9(7):2068-2075. doi: 10.1007/s12161-015-0396-z – incorporated as Chapter 3. Contributor Statement of contribution Nor Hidayah Mohd Taufek Designed study and experiments (50%) Ethics application and data collection (40%) Wrote and