Human Milk: Sampling and Measurement of Energy-Yielding Nutrients and Other Macromolecules

By Michelle McGuire

Human Milk: Sampling and Measurement of Energy-Yielding Nutrients and Other Macromolecules presents comprehensive, rigorous, state-of-the-science information on the origins, analysis, concentrations and variation in energy-yielding nutrients and other macromolecules present in human milk. The book includes information on how best to collect and store milk for determining concentrations of these important milk constituents and considers how to conduct milk composition analysis in research, clinical and resource-poor settings. Written by a group of international experts who are actively conducting research related to human milk macronutrients, each chapter also provides cutting-edge rationale for what research is still needed in this evolving field.

In addition, the book also outlines challenges and opportunities faced by clinicians, industry leaders and regulators interested in adding these components to infant foods, human milk nutrient fortifier and formula.

• Presents analytical issues and challenges
• Contains information regarding optimal milk collection and storage procedures for each milk component
• Uses a systematic treatment of common factors relating to milk composition variation (e.g., time postpartum, maternal diet)
• Provides a brief summary at the end of each chapter
• Reviews the literature related to history/discovery, analysis, isoforms, origins/transport, variability, metabolism and research gaps

• Language: English
• Publisher: Academic Press
• Release date: Nov 22, 2020
• ISBN: 9780128157077

Book preview

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Preface

In this age of year-round food availability, we often take our food supply for granted and do not really take the time to think about its fundamental importance to life. Instead, individuals and the public often focus on what not to eat rather than on the critical nature of what we should eat. But have you ever considered the basic nature of the foods we eat in terms of whether or not they have evolved to be part of the human diet? Indeed, throughout our history of hunter-gatherers to present day, we have simply co-opted and optimized natural food sources for our use. For instance, staples like rice, broccoli, and apples have been selected by farmers and families over the millennia to grow plentifully and in a particular environment. And the agriculture industry has selected and bred certain varieties, strains, and genetics to have higher yields and lower disease risk to more effectively, efficiently, and economically feed the burgeoning human population. These foods, however, did not experience natural evolutionary selection to support human health. Their goal is self-survival, reproduction, and not being eaten by humans and other animals unless that is part of their natural reproductive cycle. Indeed, most naturally occurring food sources deploy a host of natural defenses to dissuade us from eating them!

But human milk is different. Although not often considered in this way, this complex life-giving fluid is the only food that has ever truly evolved to nourish humans. Although milk produced by other mammals comes close, nothing completely compares. For instance, bovine milk needs to be fortified and substantially adulterated to support human infant growth. Indeed, with the possible exception of vitamin D, human milk is the only naturally occurring food that represents a complete and fully adequate source of vitamins, minerals, proteins, lipids, and carbohydrates needed to support life at any stage of the human lifecycle. This unique characteristic should make human milk the most studied food on the planet. Yet scientists, clinicians, public health experts, and the food industry seem to know more about what is contained in a kernel of corn than what is contained in a milliliter of human milk.

Since prehistoric times, provision of mother’s milk to newborns has been inextricably linked to the survival of the human species. The importance of breastfeeding and recommendations to address an inadequate supply are described in early writings on infant feeding; for example, in the Papyrus Ebers (c. 1550 BC) thought the first medical encyclopedia from Egypt.

To get a supply of milk in a woman’s breast for suckling a child: Warm the bones of a swordfish in oil and rub her back with it. Or: Let the woman sit cross-legged and eat fragrant bread of the soused durra, while rubbing the parts of the poppy plant

The writings of Hippocrates (460–370 BC) had little to say about infant feeding but describe his belief that suckling began in utero and accounted for the passage of the meconium and suckling strength at birth. He advised that infants be given wine diluted with water (not quite cold) to lessen the liability to convulsions. During the Roman era in his obstetrics and gynecology text, Soranus of Ephesus (98 AD–117 AD) advised on how to assess the desired quality and consistency of breast milk using observations of how a drop of milk adhered to or ran off a fingernail upright or turned downward. He also recommended that breastfeeding be avoided for two days after labor as colostrum is of poor quality, thick, and indigestible.

A small body of determined spirits fired by an unquenchable faith can alter the course of history.

Mahatma Gandhi.

Despite early historical breastfeeding guidance, the study of human milk and lactation and early infant and childhood nutrition as a scientific discipline did not fully develop until the 20th century. Two early pioneers in the field of human milk and lactation were Drs. Icie Gertrud Macy and Paul György. It is fitting that the Senior Investigator Award of the International Society for Research in Human Milk and Lactation (ISRHML) should be named after these two scientists. Icy Macy was born on a farm near Galatin Missouri in 1892. After a circuitous path balancing society expectations of women versus her natural aptitude and desire to pursue a career in science, she obtained a doctorate in physiological chemistry from Yale University in 1920 under the supervision of Lafayette Mendel (codiscoverer of vitamin A). In 1923 Dr. Macy was appointed Head of the Nutrition Research Project at the Merrill-Palmer School for Motherhood and Child Development in the Children’s Hospital of Michigan in Detroit; a position she held for 31 years. Drawing on the animal nutrition and metabolism literature, Macy designed and conducted fundamental research, which included longitudinal human and balance studies to systematically assess the metabolism of women during the reproductive cycle, nutrition, infant growth, and composition of human milk. In 1949 she published the Composition of human colostrum and milk in the American Journal of Diseases in Childhood and in 1950 with Harriet Kelly and Ralph Sloan The Composition of Milks published by the National Research Council. In 1940 Macy was appointed Chair of the Committee on Maternal and Child Feeding of the newly formed Food and Nutrition Board of the U.S. National Academies of Science. She was elected President of the American Institute of Nutrition in 1944. The social context for a career in science for women in the early 20th century was challenging. Dr. Macy served as a role model for subsequent generations working in human milk and lactation but most especially women trying to make their way in science.

Some of the founders of the International Society for Research in Human Milk and Lactation (ISRHML). Photo credit: Dr. Stephanie Atkinson.

Paul György was born in Nagyvarad, Hungary on April 7, 1893. Following in his father’s footsteps he completed his medical degree at the University of Budapest Medical School. Between 1927 and 1933 at the University of Heidelberg, and in collaboration with Professor Richard Kuhn (later Nobel laureate in Chemistry) and Dr. Th. Wagner-Jauregy, he isolated riboflavin. In 1933 he moved to the Nutrition Laboratory of Cambridge University in England as a Research Fellow where he discovered vitamin B6. As a Visiting Assistant Professor (1935) and then Associate Professor (1937) at Western Reserve University in Cleveland Ohio he isolated biotin, elucidated the structure of pyridoxine, and investigated the vitamins pantothenic acid and choline. In 1944 he moved to the University of Pennsylvania School of Medicine where he worked to the end of his life. In addition to his research, he also served as Chief of Pediatrics at various times at the Hospital of the University of Pennsylvania and Philadelphia General Hospital. It was during his time at the University of Pennsylvania, György began investigating the microbial properties of human milk in earnest, comparing the intestinal microbiota of normal breastfed infants with that of formula-fed infants. He was convinced that in addition to the important contribution of nutrients, that human milk contained molecules, now described as bioactive components, that made it unique and made it a superior form of nutrition for infants over emerging alternatives. He discovered that breastfed infants had a high prevalence of a certain variant of Lactobacillus bifidus and is well known for his discovery of L. bifidus growth factor activity in human milk and its antistaphylococcal properties.

Fast forward to the late 1970s. A group of scientists from very diverse disciplines with a strong interest in human milk and lactation began to join forces to better understand human milk composition and how its complex nature is important to maternal and infant health. These scientists were, for the most part, not trained by mentors with expertise in human milk and lactation but rather valued interdisciplinary research at a time when it was not yet fashionable. These pioneers had a strong advocate from within the U.S. National Institute of Child Health and Human Development, Thorsten A. Fjellstedt. A scientist himself, Fjellstedt recognized the growing interest in human milk and lactation, was successful in garnering some initial grant funding support, and played a pivotal role in organizing a conference in Elk Ridge Maryland in 1982 to examine methodologies in milk banking. In recognition of the need for intensive study into the methods used to analyze human milk to move the field forward, a committee was formed and a second meeting was planned to focus on laboratory research methodologies and was held in Winter Park, Colorado. This meeting, attended by 50 scientists would become the first meeting of ISRHML. The meeting also yielded the text, Human Milk Lactation: Milk Components and Methodologies in 1985 edited by Robert G. Jensen and Margaret C. Neville which became a dog-eared resource for many for decades thereafter. At this first conference and the subsequent early ISRHML conferences that followed, hours of discussion focused on methodology, a topic that might to others see unimportant. For instance, discussion topics included how does one accurately measure protein in milk, and how can we know if data coming from one lab are comparable to that originating from another. Not only did they discuss these issues, they actively debated them! This group also dug in to determine how best to collect a representative milk sample: does it matter whether it is foremilk or hindmilk, does time of day influence the constituent of interest, and does it matter if the milk is collected into plastic or glass containers? Sometimes these details were found to be unimportant, but many times they were critical aspects of being able to appropriately interpret the results of a human milk study.

We (the editors of this publication) were fortunate to experience these golden years of early human milk research while we were graduate students at the University of Illinois so many years ago. Indeed, we were mentored and trained by Dr. Mary Frances Picciano as if we were apprentices. Mary Frances (a founder of ISRHML and laureate of its Macy-György Award) and her research-partner-in-crime (and husband) Dr. John Milner demonstrated on a daily basis how to be a thorough, rigorous scientist. They introduced us to the importance of creativity in the scientific process, strength in interdisciplinary research, the power of hypothesis-driven research, and the value of methods validation and sample collection optimization. They taught us to read the literature, and more importantly, to question it. They were role models for us in using ones’ knowledge, skills, and talents in service of the common good. And they took us to ISRHML conferences!

This foundational experience really forms the underpinnings of this book. Decades after Picciano and her colleagues began seriously studying human milk composition, there remain many more questions than answers in this field of study. Furthermore, as new components of human milk are discovered and scientists attempt to study their variability in milk and importance to maternal and infant health, the same research questions reemerge: how do you collect a representative sample, and how do you accurately measure it? These questions are timeless and unfortunately are often overlooked by some contemporary researchers. Indeed, at the 2016 ISRHML meeting which was held and hosted in beautiful South Africa this topic came up in the Society’s business meeting. After a brief and lively discussion, there was an overwhelming consensus that we needed up-to-date publications on these topics. More recently, at a 2017 National Institutes of Health workshop, participants concluded that there were major knowledge gaps in our understanding of human milk composition that have led to missed opportunities to improve the health outcomes of women and children. Gaps identified included lack of standardized collection, storage, and analytical methods for many milk components and limited understanding of how milk composition changes over time or the influence of maternal and environmental factors.

As such, our objective was to work with experts around the globe in putting together his compilation of chapters specifically focused on the macronutrient and selected macromolecules in human milk. This book complements another recently published by Elsevier and coedited by Drs. Mark McGuire, Shelley McGuire, and Lars Bode which focused exclusively on complex carbohydrates and the microbiome in human milk—and is the reason that carbohydrates are not covered herein. And we are hoping that additional companion books focused on other milk components (e.g., micronutrients) emerge in the near future.

In reaching out to the authors of these chapters, we asked them to first provide an introduction and brief history of the research conducted on their nutrient or component of interest. Next, we asked authors to review factors influencing the variability of this constituent—for instance, time postpartum, time of day, maternal age, maternal diet, maternal illness, and maternal genetics. Importantly, if nothing or very little was known about what factors were related to this variability we asked our authors to state this. Extremely important to this book is methods, and you will see that each chapter provides state-of-the-science information on which types of analytical techniques are currently thought to provide the most accurate outcomes. The authors were also asked to provide normal values (and measures of variability) for each constituent and conclude their chapters with a discussion of research gaps and next steps.

We hope you agree that this consistent and critical approach throughout has resulted in the production of a reference book that will serve new and seasoned researchers alike. In addition, we anticipate that this book will provide a treasure trove of information important to allied fields such as the infant food industry and public policymakers. Indeed, there is no more important realm than advancing the science of human milk composition, because this is by definition synonymous with advancing the fundamental science of human nutrition.

Further reading

Barness and Tomarelli, 1979 Barness LA, Tomarelli RM. Paul György (1893–1976): a biographical sketch. J Nutr. 1979;109(1):19–23.

Casavale et al., 2019 Casavale KO, et al. NIH workshop on human milk composition: summary and visions. Am J Clin Nutr. 2019;110:769–779.

Jenson and Neville, 1985 Jenson RG, Neville MC. Human milk: milk components and methodologies New York: Plenum Press; 1985.

Goldman, 2020 Goldman A. History of ISRHML. https://www.isrhml.com/i4a/pages/index.cfm?pageid=3291 Last accessed August 22, 2020.

Goldman and Picciano, 2020 Goldman A., Picciano MF Macy and György. https://www.isrhml.com/files/macy-and-gyorgy.pdf Last accessed August 21, 2020.

Nichols, 2003 Nichols BL. Icie Macy and Elsie Widdowson: pioneers of child nutrition and growth. J Nutr. 2003;133:3690–3692.

Wickes, 1953 Wickes IG. A history of infant feeding Part I Primitive peoples: ancient works: Renaissance writers. Arch Dis Child. 1953;28(138):151–158.

Part I

Milk Collection and Other Methodologic Issues

Outline

Chapter 1 Collection and storage of human milk for macronutrient and macromolecule analysis—an overview

Chapter 2 Measurement of human milk production and infant milk intake—challenges and opportunities

Chapter 1

Collection and storage of human milk for macronutrient and macromolecule analysis—an overview

Meghan B. Azad¹, Stephanie Atkinson² and Donna Geddes³,    ¹1Manitoba Interdisciplinary Lactation Centre (MILC), Children’s Hospital Research Institute of Manitoba, Departments of Pediatrics and Child Health and Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada,    ²2Department of Pediatrics, McMaster University, McMaster Children’s Hospital, Hamilton, ON, Canada,    ³3School of Molecular Sciences, Faculty of Science, The University of Western Australia, Perth, WA, Australia

Abstract

Decades of research have established that breastfeeding has multiple benefits for maternal and infant health, yet much remains to be elucidated about the underlying biological mechanisms. Research in this area requires analysis of human milk and its nutritive and bioactive components, with knowledge of compositional variations that occur during the course of a single feed, diurnally, with stage of lactation, and in response to maternal diet or health status. Standardization of methods is critical to ensure accuracy and maximize comparability between studies. The optimal collection strategy will depend on the specific research question and component(s) of interest, and must consider when and how the milk is collected, as well as the processing and storage of the milk sample. In this chapter, we describe and compare different milk sampling strategies, expression methods, and storage containers, and discuss the impact of storage time and temperature on specific milk components.

Keywords

Breastfeeding; breast milk; human milk; research methods; macronutrients; specimen handling methods; infant

1.1 Introduction

Breastfeeding has many well-established short- and long-term health benefits for mothers and children. Decades of research have established that breastfed infants are protected from infections and childhood obesity, while mothers who breastfeed are protected from breast cancer and type 2 diabetes [1]XXX. These effects translate into substantial benefits to society as a whole, including higher productivity of the workforce and lower health care spending by governments [2]XXX.

Despite this large body of evidence for the beneficial effects of breastfeeding, the underlying biological mechanisms are not fully understood. Studying these mechanisms requires analysis of human milk and its nutritive and bioactive components, many of which are highly variable among women and across the stage of lactation. Standardization of milk sampling methodology is critical to ensure accuracy and allow comparability between studies. This book provides guidance for collecting human milk; measuring milk production and infant milk intake (Part I); and for analyzing human milk macronutrients (Part II) and bioactive macromolecules (Part III). Analysis of other components including micronutrients, human cells, and microbiota is reviewed elsewhere [3XXX,4]XXX.

In this chapter, we discuss the collection and storage of human milk for macronutrient and macromolecule analysis. Comparisons of milk composition among studies can be confounded by differences in milk sampling strategies and methods, which are often employed without consideration for the variability in milk composition related to the stage of lactation, diurnal patterns, changes within a feed, and maternal dietary intake from foods and supplements. The optimal strategy and method of collecting milk for research purposes will depend on the specific research question, and must consider when and how the milk is collected (Section 1.2), the method of milk expression (Section 1.3), and the handling and storage of the milk sample (Section 1.4).

1.2 Milk sampling strategies

The preferred strategy to obtain a human milk sample depends on the specific purpose of the research or clinical application (Table 1.1). Such purposes may include determining the average composition or temporal changes of human milk on a population basis; assessing the relationship between maternal nutrient intake and milk content; estimating nutrient intake by the breastfed infant, especially for monitoring in preterm infants; associating milk composition with infant health and developmental outcomes; or screening for composition and bacterial contamination of donor milk for human milk banks. This section will provide guidance in developing a strategy to obtain a representative sample of human milk in relation to the specific intended purpose and the milk component(s) of interest, considering current knowledge (and lack, thereof) of compositional variation within a single expression, throughout the day, and longitudinally over lactation. It is important to note that, for any milk sampling strategy, the natural feeding of the mother/infant dyad will be interrupted to some extent, and care must be taken to minimize inconvenience to the nursing dyad.

Table 1.1

1.2.1 Timing and volume of milk collection

As described in Parts II and III of this book, many macronutrients and macromolecules in human milk have well-defined patterns of change across lactation, from colostrum through transitional to mature milk. Protein decreases while fat and lactose increase in concentration over the first days and weeks of lactation [6,7]. Such changes in composition in early lactation may reflect the maturation of the mammary gland as suckling is initiated causing changes in the permeability of the paracellular pathway or other secretory mechanisms [6]. In contrast, many immunomodulating factors follow an opposite pattern, declining over lactation as the infant immune system matures. Some milk components are known to follow a diurnal pattern, and may also change within each feeding (or breast expression) interval. For example, total fat concentrations (and thereby energy and other fat-soluble nutrients) peak daily in the mid-morning and also increase from foremilk to hindmilk. Thus consistent and appropriate sampling procedures must be followed, and should be tailored to the specific research purpose.

Six milk sampling strategies that have been described in the literature and validated for various compositional properties of human milk are summarized in Table 1.2, adapted from a review by Miller et al. [7]. To avoid diurnal or within-feed (foremilk to hindmilk) variations, the ideal approach is to collect all milk expressed over 24 hours, and repeat this longitudinally for a population of women. While this is possible in the situation where mothers of preterm infants must pump their milk for weeks at a time until the infant is sufficiently mature to suckle at the breast, such a process is clearly disruptive to a naturally suckling mother/infant dyad. Collecting milk from one breast at each feeding (alternating breasts over 24 hours) while the infant suckles on the opposite breast is less disruptive and provides for a natural hormonally mediated milk letdown and release [8]. With this method one can pool proportional aliquots (e.g., 10% of each breast pumping) of milk over the 24-hour period, thereby obtaining a 24-hour representative sample [8]. Alternatively, for nonlipid components which vary diurnally, milk can be collected as one breast expression from a particular time of day (e.g., mid-morning) that is standardized for the collections between individuals and longitudinally within individuals (reviewed in Ballard and Morrow [10]XXX). For newly identified constituents of milk, a careful analysis should be conducted to determine all potential factors that may influence their concentration in human milk.

Table 1.2

Source: Adapted from Miller et al. [7].

When diurnal variation is not an issue and only a small volume of milk is required for the requisite analysis, the milk sampling method can be a midfeed or foremilk/hindmilk as described in Table 1.2. Midfeed sampling requires interruption of the feeding process, perhaps while the infant is being burped, while foremilk/hindmilk sampling would not interfere with the suckling process.

1.2.2 Maternal and infant health status

The health of both the mother and the infant may influence the composition of some milk constituents. For example, maternal or infant infections often stimulate an increase in concentrations of immunomodulatory components such as sIgA, IgG, and immune cells in the milk [11]. Another study showed an increase in human milk cell content in response to infant bronchiolitis accompanied by a specific cytokine profile [12]XXX. Thus, depending on the research question and milk constituent of interest, it may be critically important to assess the health of the mother and the infant as even a common cold may alter milk composition. Further, mastitis (mammary inflammation) will cause alterations in milk content and nutrient transport, so representative milk samples cannot be obtained from a mastitic breast unless the focus of the study is to understand milk composition while the mammary gland is inflamed [11].

1.2.3 Recommended milk sampling strategies for specific nutrient and macromolecule analyses

Human milk sampling strategies should be designed with consideration for the milk component(s) of interest. Table 1.3 summarizes the preferred sampling strategies for different milk components, which are briefly discussed in the following sections. Additional considerations for collecting milk to study each component are provided in Parts II and III of this book.

Table 1.3

Notes: For all nutrients, the stage of lactation is an important variable to control.

aFor some vitamins (see the text).

1.2.3.1 Lipids

The concentration of total lipids and its component parts is highly variable within a feed (increasing from foremilk to hindmilk), diurnally (varies with feeding interval [7]), across lactation (low in colostrum, rising to maximum by 2–4 weeks of lactation), and in relation to maternal diet—especially for fatty acid composition [13]. The highest lipid concentration has been reported to occur in both evening and morning, likely reflecting differences in feeding intervals [7]. When feeding occurs during the night a higher fat content in the morning milk has been observed [18]. This underlines the importance of standardizing milk collection to a consistent time of day [7]. A fully representative sample requires a complete collection of milk from a single breast or by combining aliquots using weighted averages from each total breast expression over a 24-hour period. Combining weighted average volumes of foremilk and hindmilk from a single breast expression has also been validated [13]. To minimize disruption of the breastfeeding process, collection of milk from a complete expression of one breast while allowing the infant to suckle from the other breast was considered most physiological in order to allow for the let-down reflex. For population-based studies, an alternate validated approach to minimize interference with the breastfeeding experience is removal of a single milk sample obtained 2 minutes after let-down [18]. The latter method produced similar values for total lipid compared to a 24-hour sample of complete expressions of milk from one or both breasts [19]XXX.

1.2.3.2 Protein

Consistency for the stage of lactation is required because total protein (which includes secretory immunoglobulin A and other immune proteins) declines about 30% over the first 2 weeks primarily due to a decline in the immune proteins, and reaches nadir by 4 weeks of lactation [20,21]XXX Standardization is not required for the time of day or timing within a feed, as protein concentrations do not follow a circadian pattern [22] and do not differ in foremilk versus hindmilk.

1.2.3.3 Carbohydrates

The concentration of milk lactose increases about 25% over the first month of lactation [14,20], but does not vary diurnally or within a single feed/expression. Other more complex carbohydrates including human milk oligosaccharides (HMOs) have been shown to increase or decrease over the course of lactation [15,23]XXX. It is not yet known whether HMOs vary diurnally or between foremilk and hindmilk. Thus, as for protein, milk carbohydrate analyses should account for lactation stage, but may not require standardization by time of day or timing within a feed—depending on the specific analyte of interest.

1.2.3.4 Energy

Since lipids are the major contributor to energy, the same considerations as described earlier for lipids must be followed if energy content is the major focus of analysis [22].

1.2.3.5 Vitamins

For fat-soluble vitamins such as vitamins E and A, the above considerations for lipids apply. For water-soluble vitamins such as thiamin, riboflavin, niacin, vitamin B-6, and vitamin B-12, their concentrations in human milk content often vary with maternal diet and supplement use, and with lactation stage, usually decreasing with advancing lactation [24]. Modest variation within a single feed has also been demonstrated for some water-soluble vitamins