The effect of digestive enzymes on the binding and bacteriostatic properties of lactoferrin and vitamin B12 binder in human milk

Human milk contains unsaturated lactoferrin and vitamin B12 binding protein. It has been suggested that these proteins may exert antibacterial effects in the intestine of the breast fed infant, but the effect of the intestinal environment on the antibacterial effect of these proteins has not been described. In this study human milk was treated with pepsin and trypsin and the influence of digestion on iron and vitamin B12 binding capacity, bacterial uptake of iron and vitamin B12 from milk and bacteriostatic effect was studied. Pepsin digestion had no effect on vitamin B12 binding capacity, or the ability of bacteria to take up vitamin B12, or the growth inhibitory effect on a vitamin B12 dependent strain. In contrast, trypsin digestion did not affect iron binding or bacteriostatic effects attributable to lactoferrin. The. findings support an in vivo bacteriostatic role for lactoferrin in the breast fed neonate's intestine but do not support a similar role for the vitamin B12 binding protein.     

Diet and faecal flora in the newborn: lactoferrin.

The faecal flora of breast fed babies differs from that of bottle fed babies. We have shown that the use of a whey predominant formula rather than a casein predominant one induced a faecal flora generally closer to that of breast fed babies but substantial differences remained. The whey proteins of breast milk include much more lactoferrin than is found in cows'' milk. Observations both in animals and in vitro suggest that lactoferrin could be responsible for some of these differences between bottle and breast fed babies. This study was designed to determine the effects on faecal flora of the addition of bovine lactoferrin to the diet of bottle fed babies while holding other qualities of their diet constant. As lactoferrin is an iron binding protein three test formulas were used: (a) no added iron and no added lactoferrin (basic), (b) no iron but added lactoferrin (L), and (c) added iron and lactoferrin (LF). The addition of lactoferrin had little effect upon the faecal microflora and did not move the pattern of the faecal flora in the direction of the breast fed baby. The addition of iron to the formula had more effect on the faecal flora than did lactoferrin. At day 4 it encouraged Escherichia coli and discouraged staphylococcal faecal colonisation. At day 14 the addition of iron to the formula discouraged bifidobacteria. The reasons why bovine lactoferrin was ineffective in vivo in this study are discussed.     

Antimicrobial factors in human milk

Levels of immunoglobulins, lactoferrin and lysozyme were determined in milk samples obtained from well-nourished and under-nourished Indian women at different stages of lactation. The concentration of immunoglobulins and lactoferrin was higher in colostrum than in mature milk while the lysozyme levels showed a progressive increase with the period of lactation. There were no significant differences in the levels between the two groups of women. Administration of iron did not alter either the total or percentage saturation of lactoferrin in milk. These results indicate that antibacterial factors in milk are not influenced by the nutritional status of the mother and that iron supplementation does not interfere with the bacteriostatic function of lactoferrin.     

ANTIMICROBIAL FACTORS IN HUMAN MILK

Abstract Vinodini Reddy, Bhaskaram, C., Raghuramulu, N. and Jagadeesan, V. (National Institute of Nutrition, Hyderabad, India). Antimicrobial factors in human milk. Acta Paediatr Scand, 66:229, 1977.—Levels of immunoglobulins, lactoferrin and lysozyme were determined in milk samples obtained from well-nourished and under-nourished Indian women at different stages of lactation. The concentration of immunoglobulins and lactoferrin was higher in colostrum than in mature milk while the lysozyme levels showed a progressive increase with the period of lactation. There were no significant differences in the levels between the two groups of women. Administration of iron did not alter either the total or percentage saturation of lactoferrin in milk. These results indicate that antibacterial factors in milk are not influenced by the nutritional status of the mother and that iron supplementation does not interfere with the bacteriostatic function of lactoferrin.     

CHARACTERIZATION AND PROPERTIES OF THE HUMAN AND BOVINE LACTOTRANSFERRINS EXTRACTED FROM THE FAECES OF NEWBORN INFANTS

ABSTRACT. Human lactotransferrin was characterized in the faecal extracts from breastfed babies. Quantitative determination of human copro-lactotransferrin from birth for a period of up to 3 weeks showed that the daily elimination decreased from 35 to 5 mg. The amount of copro-lactotransferrin corresponding to the endogenous secretion was calculated to be from 0.5 to 1mg per day. When a cow's milk diet supplemented by partially or completely iron-saturated human or bovine lactotransferrin was fed to the babies, the amounts of coprolactotransferrin excreted depended on the origin and on the iron saturation of the lactoransferrin. In particular, the amount of bovine copro-lactotransferrin in the faeces averaged 200 mg per day. The human and bovine copro-lactotransferrins were isolated by ion-exchange chromatography or by affinity chromatography and were still able to bind iron. The fingerprints of native human and bovine lactotransferrins hydrolysed in vitro by infant's gastric or duodenal secretions showed that both proteins were not extensively digested. This demonstrates that these lactotransferrins ingested by babies are not completely destroyed and keep their ability to bind iron, and thus may supplement the bacteriostatic effects of the endogenous lactotransferrin in the intestinal tract.     

LACTOSE AND PROTEIN ABSORPTION FROM BREAST MILK AND COW'S MILK PREPARATIONS AND ITS INFLUENCE ON THE INTESTINAL FLORA Investigations on two Infants with an Artificial Anus

Abstract. Lactose and protein absorption from breast milk and a cow's milk preparation enriched up to 7% of lactose were studied in two infants with an artificial anus applied in the ascending colon region. The concentrations of protein, lactose, glucose and galactose were measured in the fistula stools. In addition, the stools were analysed microbiologically. There were relatively high concentrations of lactose and its decomposition products and low concentrations of protein and aminonitrogen in the fistula stools when breast milk was fed. When the cow's milk formula was applied, only traces of lactose but high amounts of protein were measured. The microbiological findings are in agreement with the hypothesis that the bacterial flora of the large intestine is influenced by the lactose and protein concentrations in the intestinal content which reach the large intestine.     

ANTIBODIES TO ESCHERICHIA COLI O ANTIGENS AND THE IN-VITRO BACTERIOSTATIC PROPERTIES OF HUMAN MILK AND ITS IgA

ABSTRACT. Only a very small part of the iron-reversed bacteriostatic activity of milk against Escherichia coli , demonstrable in vitro, is due to its anti-O antibody. Most of its growth-inhibitory activity is due to another lactoferrin-dependent, non-specific system. IgA prepared from milk is bacteriostatic for E. coli in the presence of lactoferrin, if it contains O-antibody for the indicator strain and if the strain is susceptible. Susceptibility depends to some extent on virulence, since those inhibited by IgA antibody to their own O-antigens were enteropathogenic or enterotoxigenic, whereas the growth of commensal strains was inhibited only slightly or not at all.     

Iron absorption from human milk, simulated human milk, and proprietary formulas

Studies from our laboratory have shown that iron is better absorbed from human milk than from cow milk and that human milk can provide insufficient iron for infants during their first year. We compared iron availability from human milk with that from other formulas and determined the factors responsible for its superiority. Adults were fed 100 ml of human milk, simulated human milk, simulated human milk containing added lactoferrin, two commercial formulas containing iron, 12 mg/qt, and human milk that had been boiled. The simulated human milk resembled human milk in concentration of protein, fat, carbohydrate, iron, total minerals, calcium, and phosphorus. Iron 59 was added to each feeding and iron incorporation into RBCs was determined 14 days after each feeding. Percent iron absorption was highest from human milk and lowest from the commercial formulas. The simulated human milk supported a 9.0% absorption; addition of lactoferrin reduced this to 4.7%. Net iron absorption was 0.12 mg/liter from human milk and 0.40 and 0.37 mg/liter from the iron-enriched commercial formulas. Absorption of iron from boiled human milk was the same as from the unboiled milk. This study confirms the unique ability of human milk to promote iron absorption. Simple manipulation of the protein, fat, lactose, calcium, phosphorus, or lactoferrin content of proprietary milk did not reproduce the iron absorption demonstrated with human milk.     

VITAMIN E REQUIREMENTS OF PRETERM INFANTS

ABSTRACT. Differences between feeding practices in earlier investigations prompted the present study of iron and vitamin E supplementation in breast milk fed preterm infants. A new and highly sensitive technique for quantitation of alpha-tocopherol in serum was used. Studies on 34 infants with a birth weight below 2000 g or gestational age ≤35 weeks showed that supplementation with 16.5 mg tocopheryl acetate/day from 10 days of age resulted in a significantly higher haemoglobin concentration and lower reticulocyte count at 8–10 weeks than supplementation with 1.5 mg/day (p<0.05). Studies on 23 infants with a birth weight of 2000–2499 g revealed subnormal alpha-tocopherol levels in 2 of the infants given 1.5 mg tocopheryl acetate/day but there was no effect on the haemoglobin concentration at 8–10 weeks. There were no untoward effects of an early iron supplementation with 2–3 mg Fe⁺⁺ (as ferrous succinate)/kg/day. It is concluded that extra supplementation with vitamin E is advisable also in breast milk fed preterm infants. A low dosage iron supplementation from 3 weeks of age is safe.     

THE INFLUENCE OF DIETARY FACTORS ON THE VITAMIN E REQUIREMENTS OF THE LOW BIRTHWEIGHT INFANT

Jansson, L. (Department of Paediatrics, University of Lund, Malmö General Hospital, Malmö, Sweden). The influence of dietary factors on the vitamin E requirements of the low birthweight infant. Acta Paediatr Scand, Suppl. 296: 110, 1982.—The vitamin E requirements for the low birthweight (LBW) infant varies with other dietary factors such as iron and polyunsaturated fatty acids (PUFA). This interaction is caused by the influence of iron on the oxidative breakdown of lipids (lipid peroxidation). Thus the need for fortification of infant formulas with vitamin E increases if the formulas are iron fortified or have a high content of PUFA. A vitamin E/linoleic acid ratio of >1 IU/g is currently recommended. The formulas on sale in Sweden satisfies this recommendation. In breastmilk we found a vitamin E/linoleic acid ratio of s>1 IU/g in colostral and transitional milk. In mature breastmilk, however, 9 out of 24 samples showed a ratio < 1 IU/g. Thus it is possible, that the LBW infant fed mature human milk may need a larger supplement of vitamin E than the formula fed infant.     

Supplementation of milk with iron bound to lactoferrin using weanling mice: L. Effects on hematology and tissue iron

Lactoferrin is an iron-binding protein present in high concentrations in human milk. The efficacy of supplementing iron bound to lactoferrin to iron-deficient and iron-sufficient young mice was evaluated in comparison with supplementation of iron as iron chloride. Mice fed a nonsupplemented milk diet (approximately 1 mg Fe/L) for 4 weeks had a microcytic, hypochromic anemia and low tissue iron concentrations. Iron supplementation of the diet with lactoferrin-iron, or iron chloride at a level of 5 mg Fe/L prevented the anemia and resulted in tissue iron levels similar to levels found for mice fed a stock commercial diet. There was no significant difference in any of the parameters analyzed between the groups of mice receiving the two iron supplements following a diet deficient in iron. Apolactoferrin when supplemented to the diet had no negative effect on the iron status of the mice. These results show that lactoferrin may be a useful vehicle for supplementation of iron.     

Vitamin B12 deficiency in a breast fed infant.

We report the case of a 5 month old breast fed infant who presented with a history of vomiting, pallor, and failure to thrive. Investigations showed severe nutritional vitamin B12 deficiency with a megaloblastic pancytopenia. This deficiency was due to low vitamin B12 concentrations in the maternal breast milk, and subsequent investigations showed maternal pernicious anaemia. Treatment of the infant with vitamin B12 resulted in a rapid clinical and haematological improvement. This case represents an unusual presentation of pernicious anaemia.     

A SUGGESTED ROLE FOR PRECOLOSTRUM IN PRETERM AND SICK NEWBORN INFANTS

ABSTRACT. Samples of precolostrum (colostrum gravidarum), colostrum and mature milk obtained from five women during their antenatal and postnatal periods were measured for IgA, IgG, IgM, alpha-1-antitrypsin, lactoferrin, lysozyme, B, A globulin (C3) and B, E globulin (C4) by single radial immunodiffusion. Protein concentrations in precolostrum were equal to or greater than those found in colostrum obtained during the first 12-48 hours following delivery. Secretion of precolostrum is common, occurs early in the antenatal period and may often be of considerable volume. The anti-microbial proteins contained within this milk can be preserved intact by freezing. This represents an untapped pool of bacteriostatic proteins with specific activity against neonatal pathogens. We suggest that a potential protective effect against serious infection may be obtained by administering precolostrum to "at risk" infants during the first few days of life.     

An evaluation with piglets of bovine milk, hydrolyzed bovine milk, and isolated soybean proteins included in infant milk formulas. I. Effect on organ development, digestive enzyme activities, and amino acid digestibility

A study, using the piglet as a model for the human infant, was undertaken to determine the effect of the protein source of a milk formula on organ development, the activity of digestive enzymes, and the absorption of amino acids as measured at the terminal ileum and over the entire digestive tract. Three isocaloric liquid milk formulas containing equal amounts of either intact bovine milk, hydrolyzed bovine milk, or isolated soybean protein as the sole source of this nutrient and with equal levels of fat and carbohydrate were each given to six 14-day-old piglets over a 19-day period in a manner that mimicked human infant feeding practice. Following a 6-day metabolism study, the piglets were killed, their organs removed, and samples of digesta collected. The protein source of the milk-based formula did not affect (p greater than 0.05) the weight/unit body weight of the small intestine, large intestine, liver, or pancreas, but the relative weight of the kidneys was lower (p less than 0.05) for animals fed the intact bovine milk-based formula and that of the stomach was higher (p less than 0.01) for piglets receiving the isolated soybean formula. The activities of pepsin, intestinal trypsin and chymotrypsin and pancreatic chymotrypsin were not influenced (p greater than 0.05) by protein source, but piglets receiving the bovine milk-based formula had a lower level of activity (p less than 0.01) for pancreatic trypsin. The apparent ileal and fecal absorption of nitrogen was similar for the three milk-based formulas, the overall mean ileal absorption of nitrogen (+/- SE) being 89.0% (+/- 1.44). Excluding cystine, where ileal absorption was relatively lower (p less than 0.05) with the isolated soybean formula (86.7%) compared with the intact bovine milk (91.5%), protein source had little effect on the apparent absorption of essential amino acids. It was concluded that the replacement of intact cow's milk protein in human infant formulas by either hydrolyzed cow's milk or isolated soybean protein is unlikely to cause any major disturbance in the digestive process.     

Breast feeding increases concentrations of IgA in infants' urine.

To investigate the influence of breast feeding on mucosal immunity the concentrations and daily outputs of IgA and lactoferrin in urine were measured in 10 breast fed and 12 infants fed on formula milk at 6 and 12 weeks of age. The concentrations and outputs of secretory IgA in urine were significantly higher in the breast fed group by a factor of three. The secretion of IgA in urine by the breast fed infants was characteristic of the baby and was not related to the intake of IgA from breast milk. Lactoferrin concentrations were similar in the two groups at both ages. In addition to secretory IgA, two thirds of all samples contained proteins with alpha chain but no secretory component antigenic determinants. Breast feeding seems to increase the local production of secretory IgA into the urinary tract during early childhood, thus providing enhanced protection from infection.     

NITROGEN AND PROTEIN COMPONENTS OF HUMAN MILK

ABSTRACT. The true protein content of human milk is 0.9%, in well-nourished as well as malnourished mothers. Casein constitutes only about 20% of the protein nitrogen in human milk. The remaining 80 % is derived from the whey proteins, the three dominant components being α-lactalbumin, lactoferrin and secretory IgA. α-lactalbumin is a subunit of lactose synthetase. Lactoferrin is an iron-binding glycoprotein which plays a role in the defence against gastro-intestinal infections and is probably also involved in iron transport in the gut. Secretory IgA is comparatively stable at low pH; it is resistant to proteolytic enzymes and plays an essential role in the immunological defence against gastro-intestinal infections. Lysozyme is a minor component of the whey proteins and represents an active enzyme with a bactericidal effect. The nutritional and immunological significance of the marked differences with respect to the nitrogen and protein compositions of human milk and cow's milk should not be underestimated, but need further elucidation.     

Influence of the heat treatment of human milk on some of its protective constituents.

Human milk was subjected to heat treatments of graded severity and examined for its content of immunoglobulins, lactoferrin, lysozyme, vitamin B12-and folate-binder proteins, and lactoperoxidase. Holder pasteurization (62.5degrees C 30 minutes) reduced the IgA titer by 20%, and destroyed the small content of IgM and most of the lactoferrin. Lysozyme was stable to this treatment, but with an increase in temperature there was progressive destruction, to near 100% at 100degrees C. The same was broadly true of the capacity of milk to bind folic acid and potect it against bacterial uptake; with vitamin B12 the binder was more labile at 75degrees C than at 100degrees C. The milk contained no detectable lactoperoxidase.     

Lactoferrin is responsible for the fungistatic effect of human milk

Human milk has recognized anti-microbial effects and it has been repeatedly shown that breast-fed infants have fewer and less severe infections than formula-fed infants. While most studies have focused on anti-bacterial and anti-viral activities few have focused on the anti-fungal effect of human milk. Dermal and other infections caused by fungi are common in very low birth weight (VLBW) infants. Using a liquid culturing method and Candida albicans and Rhodotorula rubra as representative fungi, we studied the anti-fungal effect of human milk and certain human milk proteins. In vitro, human milk showed potent inhibitory effect on fungal growth. Most, if not all of this effect was caused by lactoferrin via its iron-binding capacity; increasing the iron content of the incubation medium abolished the inhibitory effect. In contrast, other human milk proteins with known or suggested anti-microbial effects rather increased fungal growth. Viability test and electron microscopy revealed that the growth inhibitory effect of human milk, i.e. mediated by lactoferrin, is fungistatic rather than fungicidal.