By Nick Houtman
When samples of infant digestive fluids and stool arrive in David Dallas’ Milam Hall lab, they are treated with extra care. The fluids have been carefully collected from the stomachs and small intestines of preterm infants and the stool from their diapers, all according to strict protocols. “They are very precious when we do get them,” says Dallas, an assistant professor in the College of Public Health and Human Sciences at Oregon State University.
The samples from the Oregon Health & Science University in Portland provide vital clues about how these infants digest mother’s milk. Physician-scientists at OHSU are collaborating with Dallas and his co-workers to understand how babies turn their primary source of nourishment into the building blocks of life. The results could help physicians improve the outcomes for preemies who, compared to full-term babies, tend to have more respiratory problems, infections, developmental delay and a raft of other health difficulties.
The benefits of mother’s milk are well known. Among them are reductions in lifelong risks of diabetes, obesity, food allergies and respiratory and gastrointestinal infections. Compared to infants on formula, breast-fed babies tend to have more rapid neurological development contributing to cognitive function and performance in school. The American Academy of Pediatrics and the World Health Organization recommend an exclusive diet of human milk for babies from birth to about six months of age.
Preemies at Risk
However, babies born before a full 37 weeks in the womb remain at high risk, even when mother’s milk is available. More than half a million babies — about one in 10 births — are born prematurely in the United States every year. And for those born extremely early, between 23 and 28 weeks, 1 in 4 dies in the hospital.
“Preterm babies are born with a still-developing gastrointestinal system,” says Dallas. “They have high risk for necrotizing enterocolitis, a terrible inflammatory disorder that has a high mortality rate.”
Medical intervention has increased the survival rate of babies born early, even though the most fragile may weigh barely more than a pound. And while many succeed in growing and developing well, some fail to thrive, even when they are fed mother’s milk fortified with nutrients.
“We don’t know if premature babies digest milk as well as full-term babies,” says Dallas. “They may be exposed to completely different proteins and peptides. We don’t know how digestion affects the overall protein breakdown. It’s a big black box.”
As physicians struggle to improve the prospects of these infants, researchers like Dallas offer the possibility of developing new treatments tailored to nutritional needs.
“Our goal is to grow preterm babies as well as if they had stayed inside their mothers, and we don’t always replicate that,” says Dr. Brian Scottoline, neonatologist and associate professor of pediatrics at OHSU, adjunct faculty at OSU and a collaborator with the Dallas lab. “Dave is really on the leading edge of trying to understand some of the growth problems we have with preterm babies.”
It’s quite possible that the more preterm a newborn is, the more limited they are in their ability to digest milk protein, due to the prematurity of their gastrointestinal (GI) tract. This could translate into reduced building blocks for growth.
“Furthermore, there may be important fragments of human milk proteins that have important activities in the gut. If this is true, helping babies digest dietary protein may help improve growth. Molecules derived from human milk protein could be important for development for an entire lifetime,” adds Scottoline.
The Milk Puzzle
Ironically, the complex mixture known as milk may be one of the most well-studied foods on the planet (see “Did You Know?” below). Sometimes called “liquid gold” by advocates of breastfeeding, milk is comprised of proteins, fats, sugars, antibodies and other components. They have been dissected, analyzed and subjected to batteries of experiments.
Nevertheless, advances in analytical chemistry offer unprecedented details about specific molecules in milk and in the digestive system. Milk proteins, for example, begin getting broken up into smaller pieces called peptides before the fluid even leaves the breast. Dallas and his colleagues are now able to describe these molecules down to the sequences of amino acids of which they are comprised.
Their research has already shown that in addition to complete proteins, human milk can contain between 1,000 and 2,000 peptides. The precise functions of most of them are unknown.
“And once it’s in the gut, milk is exposed to all these enzymes that break proteins down. So if you have bioactivity from those protein components in milk, it has to be from what is created inside the gut,” says Dallas. “That’s my guiding philosophy on bioactivity in nutrition. I look at what’s actually present in the intestine and the stomach.”
Working with OSU’s Mass Spectrometry Center, Dallas and his team have developed methods to identify the structures of those peptides. The researchers are also analyzing the composition of gastric and small intestine fluids that result from milk digestion in the baby’s gut – and the end product of that digestion, stool.
By comparing the structures they’ve found with previous data on peptide function, Dallas has already found peptides that are likely to have antimicrobial benefits. Others appear to boost immune function and help regulate blood pressure. It will take additional study, he adds, to confirm those results.
“We’re trying to monitor all these different peptides released from the proteins,” says Dallas. “It’s a lot of work being able to extract the peptides effectively and being able to analyze them. It’s a huge analytical challenge.”
Past human milk studies, he adds, were not always helpful for advancing infant health. Some used methods that may be irrelevant to the infant digestive system. For example, scientists have used enzymes from cheesemaking processes to digest milk proteins and determine what peptides were created. In other cases, researchers have focused on peptides that were thought to help lower blood pressure. “It’s a stretch to think that lowering blood pressure is relevant for babies,” says Dallas.
Advancing Infant Health
Dallas and Scottoline hope to gain funding for additional studies of the infant gastrointestinal system. At OHSU, Scottoline’s team is collecting samples of infant blood, urine and poop for future analysis.
The infant’s gut microbiome — the community of bacteria, fungi and viruses in the GI tract — also looms large in their concerns. Some milk peptides may feed bacteria that help fight harmful infections, while others promote disease-causing bacteria. It’s possible, says Dallas, that the way preemies digest milk could set the stage for necrotizing enterocolitis and other illnesses.
These and other issues drive work by undergraduate and graduate students and by post-doctoral scientists from around the world in the Dallas lab. Grant support comes from foundations (Gerber, Bill and Melinda Gates), companies (Glanbia) and agencies (National Institutes of Health and U.S. Department of Agriculture). A variety of projects are underway:
ANTIBODIES FOR GI TRACT HEALTH
With support from the Bill and Melinda Gates Foundation, Dallas and Scottoline collaborate in an international project to engineer antibody supplements that could confer protection from gastrointestinal illnesses. Infections by E. coli, salmonella and other microbes are a leading cause of infant death globally. The Dallas lab team, including post-doctoral scientists Jiraporn Lueangsakulthai, Baidya Sah and Bum Jin Kim, are evaluating how supplemental antibodies designed to fight specific intestinal pathogens might survive in the infant gut.
BIOACTIVE PEPTIDES AND THE MICROBIOME
With support from the NIH and the USDA, the Dallas lab, including Ph.D. student Robert Beverly and post-doctoral scholar Ningjian Liang, investigates the release of bioactive peptides from milk proteins within the preterm and term infant gut. The project also studies the effects on pathogenic and commensal bacteria, human immune cells and intestinal cell function. This work is done in conjunction with the Scottoline lab at OHSU.
ALTERNATIVES TO PASTEURIZATION
Milk donated to milk banks is pasteurized to kill microbes, but the process also destroys some useful enzymes and other proteins. With Joy Waite Cusic in the Department of Food Science and Technology, Dallas contributes to a search for alternatives — ultraviolet light, irradiation, high pressure — that can effectively sterilize milk without negative side effects.
A milk (bovine and human) molecule known as glycomacropeptide provides protection from some infectious diseases and modulates the immune system. In collaboration with Si Hong Park in the Department of Food Science and Technology, the Dallas lab (master’s students Yunyao Qu, Bryna Rackerby and Wyatt Olsen and post-doctoral student Jeewon Koh) is analyzing the impact of digestion on the survival of this molecule within adult human subjects and its effects on the human immune system and microbiome.
In a collaborative project with Adam Higgins in the College of Engineering, Scottoline and the Dallas lab are helping to develop a device to regulate glucose levels in premature infants. They aim to prevent low and high glucose levels, which can cause brain damage.
Both Dallas and Scottoline are committed to improving the lives of infants, whether born early or not. In addition to doing research, they both serve on the board of directors of the Northwest Mothers Milk Bank in Portland.
“If we didn’t think we’d be finding cool stuff, we wouldn’t get out of bed in the morning,” says Scottoline. “We’re talking about investments in people who have their whole lives.”
Did You Know?
• On average, human milk is higher in sugar and fat but lower in protein than bovine milk.
• The fat content of human milk tends to increase over the course of a single feeding.
• Breastmilk from HIV-positive women has been found to inhibit HIV transmission to infants.
• Twenty-eight milk banks in the U.S. are members of the Human Milk Bank Association of North America. Pre-term infants are at the top of the priority list for supplies.
• The Northwest Mothers Milk Bank in Portland has dispensed more than 1.6 million ounces of pasteurized human milk from 3,712 approved donors since it opened in 2013. Infants at more than 70 hospitals in the Pacific Northwest receive human milk from the bank.