Issue Date: September 29, 2008
Unraveling Breast Milk
WHEN IT COMES to feeding infants, the mantra is "breast is best." A diet of breast milk for babies is correlated with benefits including less diarrhea as well as lower incidence of diabetes or asthma when compared to formula-fed babies. But precisely how breast milk confers those advantages is unclear. Scientists know the basic ingredients of breast milk but don't fully understand how exactly they work to provide optimum nutrition for infants and protect against disease.
A better understanding of the components of human breast milk—especially its lipids and oligosaccharides—and their role in ensuring infant health could lead to improved foods and better ways to treat gastrointestinal diseases, not just for infants but perhaps also for adults. "The one thing that has evolved with humans, to nourish humans, is breast milk," says J. Bruce German, a food science professor at the University of California, Davis. "It is the ideal evolutionary model for what nourishment should be."
Human breast milk is made up of several solid components. The most abundant of those is lactose, a disaccharide that provides energy for the infant. After lactose comes lipids, which are thought to primarily deliver nutrient fat.
Milk fat typically exists in globules of varying sizes that have a triacylglycerol core surrounded by a phospholipid membrane. Beyond the basic structure, however, scientists don't know much. "For lipids and membranes the science is approximately where proteins were in the 1920s, back before researchers really had any clear understanding of the sequence and structure of individual proteins," German says.
His group is working to understand the composition and function of milk globules of varying sizes. Using laser "tweezers" to isolate single globules, they have used Raman spectroscopy to elucidate the composition of the particles (J. Agric. Food Chem. 2008, 56, 7446). The UC Davis team found that larger particles with a diameter of 5 to 10 µm do indeed have spectra consistent with triglycerides and cholesterol surrounded by phospholipids.
Smaller globules on the order of 1 µm or less, however, appear to contain few or no triglycerides. German and colleagues suggest the term "lactosomes" for these particles, to distinguish them from the traditional characterization of milk fat globules. They propose that the lactosomes are formed differently from globules and may have a function separate from simply delivering nutrient fat.
GLOBULES AND LACTOSOMES are formed at high metabolic cost to mammary cells, which sacrifice parts of their cell membrane to secrete the particles. Because mothers would not have evolved to maintain pathways that were not beneficial to infants, milk fat globules and lactosomes likely play significant roles in nutrient absorption and metabolism, German says. For example, infants tend to accumulate healthy subcutaneous fat, as opposed to the visceral fat that surrounds internal organs and is linked to diabetes and heart disease. So perhaps something about the way globules deliver fat nutrients contributes to the type of fat that develops in the body. This could have important implications for food science because common cow's milk processing operations such as homogenization break down the native globules. If cow's milk globules were left intact, perhaps children and adults who drink milk would derive additional nutritional benefits, German says.
Also, unlike typical plant oil and other animal fat storage systems, which are surrounded by a single layer of phospholipids, milk fat globules have that layer plus an additional bilayer of phospholipids and glycolipids. "It's a conspicuous excess" of material, comments German, who proposes that the role of the globules and lactosomes is to deliver not just fat but also membrane components. "When you think about it, an infant is making lots of membrane material and is turning over a lot of intestinal cells. That is probably also true for adults," German says. "People are realizing it's probably more valuable to eat phospholipids than we'd previously thought."
After lactose and lipids, free oligosaccharides are the third most plentiful solid component of human milk. Approximately 10% of the 500 calories per day that a typical mother uses to make milk are devoted to synthesizing oligosaccharides. Yet infants cannot digest the compounds. To devote so much energy to something that is not nutritious clearly indicates that the sugars must play an essential role in infant health, says Carlito B. Lebrilla, a chemistry professor at UC Davis.
Various groups, including Lebrilla's, have taken on the challenge of deciphering the full array of sugars produced in milk from humans as well as other animals, and it's a complex task. Milk oligosaccharides are composed of various linkages of five monosaccharide building blocks: D-glucose, D-galactose, N-acetylglucosamine, L-fucose, and N-acetylneuraminic acid. The resulting compounds can differ in size, charge, sequence, and abundance. Additionally, in humans the oligosaccharides secreted into milk vary depending on the mother's Lewis blood type, which is defined by the addition of fucose to specific polysaccharide chains of glycolipids and glycoproteins on red blood cells by varying fucosyltransferase enzymes.
Lebrilla and colleagues have developed a high-throughput strategy for profiling milk sugars that involves microchip liquid chromatography coupled with time-of-flight mass spectrometry (J. Agric. Food Chem. 2006, 54, 7471). The combination of techniques allows them to differentiate and quantitate not only oligosaccharides of different mass but also those with the same masses but different sugar composition and linkages. They can also work with sample sizes as small as 100 µL, an ability that is critical for investigating milk from a variety of species, including mice.
Although previous estimates of the number of different human milk oligosaccharides numbered in the thousands, Lebrilla and coworkers have found that humans actually put into milk only about 200 different oligosaccharides that range in size from 2 to 32 residues. One individual's milk can contain anywhere from a few dozen to more than a hundred different oligosaccharides. And although some researchers have reported that the number of oligosaccharides varies over time as the infant grows, recent work by Lebrilla and coworkers shows that that is true only for minor oligosaccharide components of human milk???the quantity of the major components stays relatively constant over a three-month period (J. Agric. Food Chem. 2008, 56, 618).
Lebrilla's work to illuminate the oligosaccharide composition of milk is now being used to test what researchers believe is a major role of oligosaccharides in milk: As a food source for particular bacteria, they coax health-promoting microbes to colonize the digestive tracts of infants. The sugars in human breast milk, for example, appeal to certain strains of bacteria, called bifidobacteria, that can colonize the gut and appear to be important for the health of infants. Well-established colonies of bifidobacteria can prevent pathogens such as harmful strains of Escherichia coli from getting a foothold.
Lebrilla is working with UC Davis colleagues German and David A. Mills, a professor and microbiologist, to delineate exactly which bacteria ferment which oligosaccharides (J. Agric. Food Chem. 2007, 55, 8914). The results indicate that mothers have evolved to produce breast milk with the correct components to recruit specific gut-dwelling bacteria that promote rather than harm their infants' health.
One area in which this knowledge might be useful is in feeding premature infants, who are particularly susceptible to a gastrointestinal disease called necrotizing enterocolitis (NEC) which essentially destroys the bowel. Approximately 10% of premature infants get NEC, and of those, 40% die. One hypothesis about the source of the disease is that infants sequestered in intensive care units are not seeded properly with beneficial bacteria, leaving them vulnerable to hospital-acquired organisms. A solution might be to feed preemies a cocktail of prebiotics—food for beneficial bacteria, such as specific breast milk oligosaccharides—and probiotics—the desired bacteria—to promote the development of healthy gut flora and protect effect against disease.
To that end, the UC Davis scientists are working with Mark A. Underwood, a professor of clinical pediatrics at UC Davis' Children's Hospital, on a clinical trial involving feeding infants various oligosaccharides and their corresponding bacteria to observe the impact on intestinal microflora as well as infant growth and overall health. The researchers also believe that feeding targeted prebiotic sugars and probiotic bacteria could be used to treat gastrointestinal diseases in adults, by providing a means to restore microbial balance in the digestive tract. It's an approach that is similar in concept to that promoted by current products such as yogurts that contain probiotic bacteria, but is informed by the evolutionary interplay between mothers, the milk oligosaccharides they produce, and the bacteria that grow in healthy infants, Mills emphasizes.
Formula for healthy infants could also be redesigned to include oligosaccharides that promote the growth of bacteria found in breast-fed babies; right now, formula-fed infants tend to develop colonies of more "adultlike" intestinal microbes in their digestive tracts. Researchers hypothesize that the different microbes might be one reason rates of diarrhea are higher in babies fed formula rather than breast milk.
The role of oligosaccharides in infant health is not, however, limited to feeding bacteria. The epithelial cells that line the intestine are decorated with glycans that mediate communication with the extracellular environment, facilitating cell-cell communication as well as binding to signaling agents. Intestinal pathogens frequently bind to those glycans as part of pathogenesis. Free oligosaccharides can, therefore, serve as decoys or inhibitors to prevent pathogen binding to intestinal cells. Lebrilla and colleagues have recently demonstrated that human milk oligosaccharides interfere with human immunodeficiency virus binding to dendritic cells, helping to explain why a majority of infants breast-fed by HIV-positive mothers do not develop the disease (Br. J. Nutr., DOI: 10.1017/S0007114508025804).
Breast milk oligosaccharides can also defend against bacterial infections. David S. Newburg, a professor of pediatrics at Harvard Medical School, in collaboration with Guillermo M. Ruiz-Palacios of Mexico's National Institute of Medical Science & Nutrition, and Ardythe L. Morrow, director of the Center for Epidemiology & Biostatistics at Cincinnati Children's Hospital Medical Center, has found that α1,2-linked fucosylated oligosaccharides in human breast milk can prevent E. coli stable toxin, Campylobacter jejuni, and Vibrio cholerae—all of which cause diarrhea—from binding to cellular targets, thus inhibiting pathogenesis (Annu. Rev. Nutr. 2005, 25, 37).
Newburg is working on his own procedures for identifying and characterizing milk oligosaccharides; his latest methods use capillary electrophoresis to elucidate acidic oligosaccharides (Anal. Biochem. 2007, 370, 206). Milk is a difficult substance to work with analytically, Newburg says, noting that in addition to the molecular diversity, there are different phases of solution. "It's a very complex and messy material to work with," he says.
Newburg's lab is continuing to investigate the relationships between milk oligosaccharides and disease. One project aims to elucidate the mechanism by which breast milk oligosaccharides prevent respiratory disease. Newburg and colleagues are also looking at the interrelationships between milk oligosaccharides and intestinal bacteria. Newburg, Ruiz-Palacios, and Morrow have started a Massachusetts-based company, Glycosyn, to engineer bacteria and yeast to synthesize human milk oligosaccharides to provide material for prebiotics or infant formula.
Going beyond free oligosaccharides, glycans attached to fat globules or proteins in breast milk may also play a role in disease prevention. Preliminary work in German's lab, in collaboration with UC Davis applied science professor Atul N. Parikh, indicates that lactosomes can bind to cholera toxin, likely through a glycolipid on the surface of the lactosome.
In another example, the milk glycoprotein lactadherin inhibits pathogenesis of rotavirus, which is the leading cause of severe diarrhea in young children. Lactadherin is glycosylated with a sialic acid; when the sugar is removed from the protein, the protein loses its ability to inhibit the pathogen. A study by Newburg and coworkers also demonstrated that a lower incidence of rotavirus-induced diarrhea in infants correlated to higher concentrations of lactadherin in their respective mother's milk (Lancet 1998, 351, 1160).
ALL BUT ONE of the 10 most abundant proteins in human milk are glycosylated. Lebrilla and colleagues are now developing techniques to map the site-specific glycosylation patterns of all milk glycoproteins. "Say you can have three types of glycosylation sites on a protein. If each of those can have 10 different glycans, then you have 1,000 different glycoforms for just one protein," Lebrilla says to illustrate the magnitude of the task.
Whereas scientists interested in studying protein glycosylation traditionally had to denature, reduce, alkylate, and digest proteins before separating the components and using techniques like mass spectrometry to identify the sugars, Lebrilla and coworkers use the enzyme pronase to cleave all the peptide bonds except at glycosylation sites, then use mass spectrometry to identify the remaining peptides and sugars (Anal. Chem. 2003, 75, 5628). It takes them a day and half to accomplish what formerly took years, Lebrilla notes.
The group has focused on lactoferrin and has found that the specific glycosylation pattern of the protein varies over time. In a study of five mothers, lactoferrin was glycosylated for the first 10 days of lactation, and then the sugars mostly disappeared. Ten days is also the point at which bifidobacteria are generally well established in the intestines, Lebrilla notes. He suggests that the mother delivers glycosylated lactoferrin to play a protective role against pathogens while the infant builds up intestinal microbes. Once the bifidobacteria are well established, lactoferrin—easier to digest when not glycosylated—becomes a protein nutrient.
Lactoferrin delivers iron to the infant as well. Breast milk also contains other essential minerals, vitamins, antibodies, hormones, growth factors, signaling molecules, and white blood cells. And it can contain environmental contaminants such as perchlorate that are passed from the mother to the infant.
Much still remains to be understood about how many of milk's natural components are synthesized and delivered, how synthesis is controlled, and the effects of the mother's diet on the final product. "It is a remarkable fluid," German emphasizes. "It's extremely embarrassing how little we still know about it."
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