Human milk macro- and trace elements in Bangladesh and their response to acute maternal supplementation

Authors: HAMPEL, DANIELA - University Of California, Davis; ISLAM, MUNIRUL - International Centre For Diarrhoeal Disease Research; Shahab-Ferdows, Setareh; Allen, Lindsay

Submitted to: Current Developments in Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/20/2024
Publication Date: 6/29/2024
Citation: Hampel, D., Islam, M.M., Shahab-Ferdows, S., Allen, L.H. 2024. Human milk macro- and trace elements in Bangladesh and their response to acute maternal supplementation. Current Developments in Nutrition. 8(2). Article 103736. https://doi.org/10.1016/j.cdnut.2024.103736.
DOI: https://doi.org/10.1016/j.cdnut.2024.103736

Interpretive Summary: Exclusively breastfeeding (EBF) infant receive their needed nutrients, including macro- and trace-elements, solely from human milk. Hence, accurate analysis to assess risk of deficiencies of these compounds, and evaluate the effects of interventions, is vital. Even recent methods for macro- and trace-elements report the need for multiple approaches and for several milliliters, drawbacks for obtaining timely results and problematic if there is only little sample volume available. These disadvantages now have been overcome with our approach for simultaneous analysis of macro- and trace-elements reported here. We optimized and validated the simultaneous analysis of 13 macro- and trace-elements by inductively-coupled plasma-mass spectrometry. 100-600µL of the milk were microwave digested with = 1.5mL nitric acid (70%). The digest was diluted to 5% final acid concentration. He-Kinetic Energy Discrimination (KED; Na, K, P, Ca, Mg, Fe, Cu, Zn, Cr, Mo) and O2-Dynamic Reaction Cell (DRC; As, Mn, Se) modes minimized remaining polyatomic interferences. Accuracy (NIST SRM 1869 infant formula; n=15, 4 weeks) varied from 92.5-103.3% (CV: 3.0-5.6%). Inter-day variation of a HM-pool (n=20, 3 weeks) varied between 4.1-8.5% for most elements; Cr, Mo, Mn (all<5ppb) varied up to 25%. Analyzing HM from 18 Bangladeshi mothers (2-4 months postpartum; day 1 = baseline, n=17; day 2/3 = supplementation, n=21 each) revealed higher concentrations for P, Ca, and Zn post-supplementation (p<0.05, Friedman’s Chi-Square Test). Na, Mg, Zn, and Se had the highest number of samples (>80%) with concentrations below the Adequate Intake. Thus, our method offers simultaneous and reproducible analysis of macro- and trace-elements with concentrations ranging over 6 orders of magnitude, without the need for separate methods and sample preparations, and requiring only sub-milliliter amounts of HM. Additional elements may be included after optimization and validation. The results from Bangladeshi HM samples indicate selective supplementation effects and concerningly low concentrations for some elements, which could adversely affect the EBF infant.

Technical Abstract: Background: Adequate concentrations of human milk (HM) nutrients, including macro- and trace-elements, are essential for healthy growth and development of exclusively breastfed (EBF) infants. To monitor potential risk of deficiencies, and evaluate the effects of interventions such as supplementation, accurate analysis of HM is crucial. Even recent methods reporting on HM macro- and/or trace-elements describe multiple methodological approaches and the need for several milliliters, drawbacks for obtaining timely results and a problem if there is limited availability of sample volume. These disadvantages now have been overcome with our comprehensive method for simultaneous analysis of macro- and trace-elements reported here. Results: 13 macro- and trace-elements were optimized and validated for simultaneous analysis by inductively-coupled plasma-mass spectrometry. 100-600µL HM were microwave digested with = 1.5mL HNO3 (70%). The digest was diluted to 5% final acid concentration. He-Kinetic Energy Discrimination (KED; Na, K, P, Ca, Mg, Fe, Cu, Zn, Cr, Mo) and O2-Dynamic Reaction Cell (DRC; As, Mn, Se) modes minimized remaining interferences. Accuracy (NIST SRM 1869 infant formula; n=15, 4 weeks) varied from 92.5-103.3% (CV: 3.0-5.6%). Inter-day variation of a HM-pool (n=20, 3 weeks) varied between 4.1-8.5% for most elements; Cr, Mo, Mn (all<5ppb) had higher variation, up to 25%. Analyzing HM from 18 Bangladeshi mothers (2-4 months postpartum; day 1 = baseline, n=17; day 2/3 = supplementation, n=21 each) revealed higher concentrations for P, Ca, and Zn post-supplementation (p<0.05, Friedman’s Chi-Square Test). Na, Mg, Zn, and Se had the highest number of samples (>80%) with concentrations below the Adequate Intake. Significance: Our method allows for simultaneous and reproducible analysis of macro- and trace-elements with concentrations ranging over 6 orders of magnitude, without the need for separate analytics and sample preparations, and requiring only sub-milliliter amounts of HM. Additional elements may be included after optimization and validation. The results from Bangladeshi HM samples indicate selective supplementation effects and concerningly low concentrations for some elements, which could adversely affect the EBF infant. Background (80-100 words): Adequate concentrations of human milk (HM) nutrients, including macro- and trace-elements, are essential for healthy growth and development of exclusively breastfed (EBF) infants. To monitor potential risk of deficiencies, and evaluate the effects of interventions such as supplementation, accurate analysis of HM is crucial. Even recent methods reporting on HM macro- and/or trace-elements describe multiple methodological approaches and the need for several milliliters, drawbacks for obtaining timely results and a problem if there is limited availability of sample volume. These disadvantages now have been overcome with our comprehensive method for simultaneous analysis of macro- and trace-elements reported here. Results (125-150 words): 13 macro- and trace-elements were optimized and validated for simultaneous analysis by inductively-coupled plasma-mass spectrometry. 100-600µL HM were microwave digested with = 1.5mL HNO3 (70%). The digest was diluted to 5% final acid concentration. He-Kinetic Energy Discrimination (KED; Na, K, P, Ca, Mg, Fe, Cu, Zn, Cr, Mo) and O2-Dynamic Reaction Cell (DRC; As, Mn, Se) modes minimized remaining interferences. Accuracy (NIST SRM 1869 infant formula; n=15, 4 weeks) varied from 92.5-103.3% (CV: 3.0-5.6%). Inter-day variation of a HM-pool (n=20, 3 weeks) varied between 4.1-8.5% for most elements; Cr, Mo, Mn (all<5ppb) had higher variation, up to 25%. Analyzing HM from 18 Bangladeshi mothers (2-4 months postpartum; day 1 = baseline, n=17; day 2/3 = supplementation, n=21 each) revealed higher concentrations for P, Ca, and Zn post-supplementation (p<0