Bioactive factors in human milk.

This article reviews the bioactive components of human milk. Special emphasis is given to immune and nonimmune protective function of major and minor nutrients in human milk. Immune modulating components, such as cytokines, nucleotides, hormones, and growth factors, are discussed. Milk enzymes with digestive function in the newborn are reviewed.

Structural and functional aspects of three major glycoproteins of the human milk fat globule membrane.

The MUC1 mucin, lactadherin, and butyrophilin are 3 major components of the human milk fat globule membrane. The mucin inhibits binding of S-fimbriated Escherichia coli to buccal epithelial cells, and lactadherin prevents symptomatic rotavirus infection in breast-fed infants. Butyrophilin has been suggested to be a structural component of the human milk fat globule (HMFG) membrane and to have receptor functions, but has no known anti-infective activity. These HMFG glycoproteins also are present in skimmed milk, possibly associated with phospholipid micelles, while mucin is also in a soluble form. Mucin and lactadherin resist digestion in the stomach of milk-fed infants, while butyrophilin is rapidly degraded. The MUC1 mucin is an extended rod-like structure forming part of the glycocalyx on the surface of many epithelial cells and membranes of milk, and may act as a decoy for binding of infective agents. The extracellular segment of butyrophilin has homology to Ig superfamily receptors and an intracellular domain with homology to developmentally regulated proteins. Lactadherin is a laterally mobile cell adhesion molecule that interacts with integrins and has a novel means of membrane-association involving specific binding to phosphatidylserine. The structural and functional aspects of these glycoproteins are discussed with regard to their role in human milk for breast-fed infants.

Long-chain polyunsaturated fatty acids (LC-PUFA) during early development: contribution of milk LC-PUFA to accretion rates varies among organs.

Long-chain polyunsaturated fatty acids (LC-PUFA) accretion (essential for growth and neural development) was studied from late fetal throughout weaning age in the ferret, a species with maternal LC-PUFA sufficiency during pregnancy and lactation. The data show that a) accretion rate of LC-PUFA is rapid during early postnatal development, b) milk LC-PUFA decrease during lactation, c) adipose tissue LC-PUFA level is directly related to milk LC-PUFA level, while accretion in brain and liver exceeds dietary intake, d) accretion of arachidonic acid occurs earlier than docosahexaenoic acid, suggesting earlier development of n6-fatty acid endogenous synthesis.

Gastric proteolysis in preterm infants fed mother's milk or formula.

Gastric proteolysis is assumed to be low in the newborn (Britton & Koldovsky 1989). Postprandial pepsin output is significantly lower in preterm infants than adults, 589 vs. 3352U/kg, respectively (Armand et al. 1995, 1996). We now report on gastric proteolysis in preterm infants (gestation age, 29 weeks; postnatal age, 5-6 weeks) gavage-fed mother's milk or preemie formula. The data show that a) the nonprotein component is higher in human milk than formula, b) net proteolysis amounts to 15% of protein, c) gastric proteolysis is lower than lipolysis and, contrary to the latter, is not enhanced by milk feeding (Armand et al. 1996). We suggest that stomach pH, enzyme output, and food structure are the reasons for differences in gastric digestion of protein and fat in infants.

Protective function of human milk: the milk fat globule.

Human milk contains many components that protect the newborn against infection at a time when the infant's own defense mechanisms are poorly developed. Fat is one of the major nutrients in human milk. The fat is contained within milk fat globules composed of a core of triglyceride and a membrane consisting of phospholipids, cholesterol, proteins, and glycoproteins. Both the membrane and the core components can provide protection against microorganisms. The major protective membrane glycoproteins, mucin, and lactadherin are resistant to conditions in the newborn's stomach and maintain their structure and function even at low pH and in the presence of the proteolytic enzyme pepsin. The core triglycerides upon hydrolysis by digestive lipases (especially gastric lipase, which is well developed in the newborn) produce free fatty acids and monoglycerides, amphiphylic substances able to lyse enveloped viruses, bacteria, and protozoa. Therefore, in addition to its nutritional value, the fat in human milk has a major protective function.

Milk fat globule glycoproteins in human milk and in gastric aspirates of mother's milk-fed preterm infants.

Human milk fat globule (HMFG) glycoproteins can prevent infections by microorganisms in breast-fed infants; the MUC-1 mucin inhibits binding of S-fimbriated Escherichia coli to buccal mucosa, and lactadherin may prevent symptomatic rotavirus infections. In this study, the survival of these HMFG glycoproteins in the stomach of human milk-fed preterm infants (gestational age = 27.5 +/- 0.4 wk) was assessed, and levels in their mothers' milk determined, using specific RIAs. Butyrophilin, a major component of HMFG membrane that has no demonstrated antimicrobial activity, was studied for comparison. The levels of mucin, lactadherin, and butyrophilin in 41 milk samples of 20 mothers were 729 +/- 75, 93 +/- 10, and 41 +/- 3 microg/mL, respectively. Mucin and lactadherin were significantly higher in early milk samples (<15 d postpartum) than in later milk samples (15-90 d postpartum), whereas butyrophilin showed no such difference. Significant amounts of mucin and lactadherin were found in almost all gastric aspirates of human milk-fed infants, even 4 h after feeding (mucin, 270 +/- 30 microg/mL; lactadherin, 23.2 +/- 4.4 microg/mL), whereas butyrophilin was rapidly degraded in the majority of aspirates. Western blot analysis demonstrated that the immunoreactive mucin, lactadherin, and butyrophilin in the milk-fed gastric aspirates had the expected native molecular weights. Mucin and lactadherin survived at all gastric pH values, whereas butyrophilin was found only at pH > 4. Neither lactadherin nor butyrophilin were detected in gastric aspirates of formula-fed infants (gestational age = 27.8 +/- 0.5 wk), whereas the very low level of mucin (9.1 +/- 1.1 microg/mL) in this group is presumably cross-reacting gastric mucin. These results demonstrate that two HMFG glycoproteins implicated in prevention of infection, MUC-1 mucin and lactadherin, survive and maintain their integrity in the stomachs of human milk-fed preterm infants.

Long-chain polyunsaturated fatty acids.

Long-chain polyunsaturated fatty acids (LC-PUFA) are essential for normal development. Fetal accretion of LC-PUFA occurs during the last trimester of gestation; therefore, premature infants are born with minimal LC-PUFA reserves. Recent studies indicate that the newborn can synthesize LC-PUFA from essential fatty acid precursors; however, the extent of de novo synthesis remains to be established. Postnatally, human milk provides LC-PUFA to the newborn. Maternal LC-PUFA reserves depend upon diet and can be improved by supplementation of docosahexaenoic acid and arachidonic acid during pregnancy and lactation. This in turn affects fetal LC-PUFA accretion and postnatal provision through mother's milk. Supplementation of formula-fed preterm or full-term infants with docosahexaenoic acid and arachidonic acid leads to plasma and red blood cell LC-PUFA levels similar to those of breast-fed infants. The higher blood and presumably tissue levels of LC-PUFA following supplementation lead, however, to only temporary functional benefits.

Glycoproteins of the human milk fat globule in the protection of the breast-fed infant against infections.

Nonimmunological components in human milk can protect breast-fed infants against infection by microorganisms. The structural and functional characteristics of four such components are discussed. The mucin inhibits binding of S-fimbriated Escherichia coli to bucal epithelial cells; lactadherin prevents symptomatic rotavirus-induced infection; glycoaminoglycans inhibit binding of human immunodeficiency virus gp120 to its host cell CD4 receptor, and oligosaccharides provide protection against several pathogens and their toxins.

Protective function of proteins and lipids in human milk.

Human milk provides the infant with protection against infectious diseases. This protection is conferred through several mechanisms: specific antibody targeted protection against pathogens in the infant's environment (through milk IgA, IgG, and IgM) and broad-spectrum, nonspecific protection provided through several distinct mechanisms. These are: bactericidal effects (lactoferrin), bacteriostatic action (lactoferrin, lysozyme), lysis of microorganisms (lysozyme), antiviral effects (lactoferrin, products of milk fat digestion), antiprotozoan activity (free fatty acids produced during gastric and intestinal digestion of milk fat), and ligand action (inhibition of Helicobacter pylori adhesion to gastric mucosa by kappa-casein). In addition to these protective functions of the proteins and lipids of human milk, several enzymes present in human milk might provide protection by generating components that are bactericidal (bile salt dependent lipase, peroxidase), prevent inflammatory reactions (platelet-activating factor acetylhydrolase), or protect the integrity of milk proteins (antiproteases).

Early and late effects of breast-feeding: does breast-feeding really matter?

Breast-feeding protects the newborn against infectious diseases in developing as well as in industrialized countries. Protection is conferred against gastrointestinal and respiratory tract diseases as well as against otitis media. This protection provided by specific (antibody dependent) and broad, nonspecific protective factors in human milk (proteins, glycoproteins, and lipids) is associated with lower global morbidity and mortality of breast-fed infants as compared with formula-fed infants. While protection against diseases that develop later in life, such as insulin-dependent diabetes mellitus, inflammatory bowel disease, and childhood cancer, has been reported, well-planned prospective studies are essential in order to confirm these observations. Similar studies are essential in order to ascertain the small but consistently reported higher cognitive ability of breast-fed infants.

Molecular cloning of the bile salt-dependent lipase of ferret lactating mammary gland: an overview of functional residues.

Ferret lactating mammary gland bile salt-dependent lipase (BSDL, EC 3.1.1.-) has been cloned by RT-PCR. The open reading frame consists of 1869 nucleotides which encode 623 amino acids of the functional enzyme. When compared to other species, the greatest homology is observed between residues 1 and 484, with little or no homology at the C-terminal end where seven repeated segments of similar sequence are located. Ferret mammary gland BSDL retains residues involved in the active site and the tentative heparin binding site at similar positions in comparison to other milk or pancreatic BSDL. Other important items, such as binding peptide to chaperone molecular, phosphorylation site(s) or bile salt binding sites, were also tentatively located in well conserved regions of seven available BSDL sequences.

Effect of pasteurization on long chain polyunsaturated fatty acid levels and enzyme activities of human milk.

Milk fatty acids, including the polyunsaturated long chain fatty acids essential for retinal function and brain development, are not affected by pasteurization (62.5 degrees C for 30 min). Milk lipases are completely destroyed by pasteurization, whereas amylase lost only 15% of initial activity. Thus, certain bioactive components are stable to pasteurization of donor milk and can benefit the recipient infants.

Gastric lipase and pepsin activities in the developing ferret: nonparallel development of the two gastric digestive enzymes.

BACKGROUND: Gastric lipase has an important compensatory function in neonatal fat digestion. The activity level of pepsin and its role in protein digestion is less well understood. We have, therefore, studied the ontogeny of lipase and pepsin in the ferret, a species with a neonatal fat digestion pattern similar to that of humans. METHODS: Gastric lipase and pepsin activities were quantified from the late fetal period throughout lactation, and were compared with those of the adult. RESULTS: The data show earlier ontogeny and much more rapid rise of lipase activity than of pepsin. Lipase activity was present during the last week of fetal development, whereas pepsin was detected only postnatally. Lipase activity was 72.8% +/- 14.2% and 153% +/- 9.95% and pepsin activity was 11.6% +/- 1.3% and 30.1% +/- 1.3% of the adult level at 2 and 4 wk of age, respectively. CONCLUSIONS: We conclude that lipase activity develops early and exceeds adult activity during the suckling period, when fat intake is very high. The low pepsin activity and high postprandial pH probably limit gastric proteolysis, thereby contributing to the structural and functional stability of milk proteins, many with protective or bioactive function in the gastrointestinal tract of the newborn.

Digestive enzymes in human milk: stability at suboptimal storage temperatures.

BACKGROUND: Women who return to work outside of the home while still breastfeeding must often store the expressed milk at less than optimal temperatures. Human milk provides digestive enzymes (amylase and lipase) that compensate in the newborn for immature pancreatic function. METHODS: We have assessed the stability of amylase and bile salt-dependent lipase after storage for 1-24 h at 15, 25, and 38 degrees C. RESULTS: Both enzymes were stable at 15 and 25 degrees C for 24 h, whereas at 38 degrees C there was a 15 and 20% decrease in lipase and amylase activity, respectively. The stability of milk lipoprotein lipase was also tested. This very labile enzyme was more stable in milk than previously reported for blood and tissues, i.e., 20 and 50% decrease in activity after storage at 15 or 25 degrees C for 24 h, respectively. A two-unit drop in milk pH by 24 h of storage would not affect the activity of digestive enzymes, which are stable at pH > 3.5. CONCLUSIONS: We conclude that milk provides the same compensatory digestive activity after short-term storage, even at relatively high temperature, as when fed fresh to the infant.

Effect of human milk or formula on gastric function and fat digestion in the premature infant.

The effect of diet, human milk or formula, on gastric function (lipase and pepsin activity, pH, and volume) and intragastric digestion of fat was assessed in 28 appropriate for gestational age preterm infants (gestational age, 28.9 +/- 1.4, 29.1 +/- 0.9, 29.5 +/- 0.6 wk; birth weight, 1.00 +/- 0.14 to 1.18 +/- 0.07 kg). The infants were fed either human milk (n = 11), SMA Super Preemie formula (n = 9), or Similac, Special Care formula (n = 8). Fasting and postprandial activity of digestive enzymes, pH, and gastric volume (measured before or during 50 min after gavage feeding) did not differ as a function of diet among the three groups of infants. Gastric lipase output, 23.1 +/- 5.1, 28.3 +/- 6.6, and 22.5 +/- 6.4 (U/kg of body weight) in human milk-, SMA SP-, or Similac SC-fed infants was comparable to the gastric lipase output of healthy adults fed a high fat diet (22.6 +/- 3.0). Pepsin output was, however, significantly lower (597 +/- 77, 743 +/- 97, and 639 +/- 142 U/kg of body weight) in human milk-, SMA SP-, and Similac SC-fed infants) than in healthy adults (3352 +/- 753 U/kg). The hydrolysis of dietary fat was 1.7-2.5-fold higher (p < 0.01) in human milk-fed infants than in infants fed either formula. We conclude that differences in type of feeding, i.e. different fatty acid profiles (long chain or medium chain triglycerides), different emulsions (natural or artificial), and different fat particle sizes do not affect the level of activity of gastric enzymes. However, the triglyceride within milk fat globules appears to be more accessible to gastric lipase than that within formula fat particles. We suggest that the contribution of gastric lipase to overall fat digestion might be greater in the newborn (a period of pancreatic insufficiency) than in the adult.

Breastfeeding: Unraveling the Mysteries of Mother's Milk.

Most of the major progress in understanding the unique and complex features of human breast milk has emerged in just the past 2 decades. Since the late 1970s, key research has examined such aspects as the composition of breast milk, effects of maternal and environmental factors on human milk, and the effect of human milk on the infant, including the protection against disease that breast milk can confer on the newborn. The composition of human breast milk includes growth factors, hormones, enzymes, and other substances that are immune-protective and foster proper growth and nutrition in the newborn. Research suggests that lactation is robust and that a mother's breast milk is adequate in essential nutrients, even when her own nutrition is inadequate. Mature breast milk usually has constant levels of about 7g/dL carbohydrate and about 0.9g/dL proteins. But the composition of fats essential for neonatal growth, brain development, and retinal function varies according to a woman's intake, the length of gestation, and the period of lactation. Vitamins and minerals also vary according to maternal intake. But even when these nutrients are lower in breast milk than in formulas, their higher bioactivity and bioavailability more nearly meet the complete needs of neonates than do even the best infant formulas. Also, in many instances human milk components compensate for immature function, such as a neonate's inability to produce certain digestive enzymes, immunoglobulin A (IgA), taurine, nucleotides, and long-chain polyunsaturated fatty acids.