THE EFFECT OF DIGESTIVE ENZYMES ON THE BINDING AND BACTERIOSTATIC PROPERTIES OF LACTOFERRIN AND VITAMIN B12 BINDER IN HUMAN MILK

Abstract. Samson, R. R., Mirtle, C. and McClelland, D. B. L. (University Department of Therapeutics and Clinical Pharmacology, The Royal Infirmary, Edinburgh, Scotland). The effect of digestive enzymes on the binding and bacteriostatic properties of lactoferrin and vitamin B12 binder in human milk. Acta Paediatr Scand, 69:517, 1980.—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 dependant strain. In contrast, pepsin digestion (or low pH alone) released iron from milk and abolished its bacteriostatic effect. Trypsin digestion slightly reduced the molecular size of the vitamin B12 binding protein without releasing free vitamin B12; the bacteriostatic effect on a vitamin B12 dependant organism was, however, abolished. 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.     

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.     

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.     

Expression,characterization, and biologic activity of recombinant human lactoferrin in rice

BACKGROUND: Lactoferrin has been suggested to have many biologic activities, such as facilitating iron absorption and having antimicrobial and antiinflammatory effects. In humans, several of these activities are likely to only be facilitated by human lactoferrin because they depend on the binding of human lactoferrin to specific receptors. Rice may be a useful vehicle to introduce recombinant human lactoferrin to infant foods because it has low allergenicity and is likely to be safer than using microorganisms or transgenic animals. METHODS: Recombinant human lactoferrin was expressed in the rice cell culture system, and its biologic activity was assessed by iron-binding and -releasing properties, antimicrobial activity, and binding and uptake to Caco-2 cells. The authors also compared the stability of recombinant and native human lactoferrins against heat, low pH, and in vitro digestion. RESULTS: Biologic activity of rice-expressed recombinant human lactoferrin was similar to that of native human lactoferrin. Heat-treated proteins retained their functional activities except with severe treatment at 100 degrees C for 8 seconds, which disturbed the iron-binding capacity of recombinant human lactoferrin. Both types of proteins retained their functional activities between pH 2 and 7.4. After in vitro digestion, 50% of both proteins were detectable by enzyme linked immunosorbent assay. The remaining native and recombinant lactoferrins retained antimicrobial and Caco-2 binding and uptake activities. CONCLUSIONS: The results indicate recombinant human lactoferrin has stability similar to native human lactoferrin when exposed to thermal treatment, pH treatment, and in vitro digestion, suggesting it may be active when added to infant formula.     

Diet and faecal flora in the newborn: iron.

The faecal flora of a baby receiving a modern infant formula is substantially different from that of a breast fed baby. This difference is a little less when whey based formulas are used. The addition of bovine lactoferrin has no effect and there is some evidence that the presence of added iron in a formula moved the faecal flora further away from that of a breast fed baby. the iron content of currently used infant formulas is much higher than that of breast milk. The effect of the addition of iron to both whey and casein based formulas on the faecal flora was examined in further detail. Faecal flora were examined at 14 days of age in 33 babies receiving a whey formula fortified with iron, 29 babies receiving a whey formula without iron, 29 babies receiving a casein formula fortified with iron, and 24 babies receiving casein formula without iron. Subsequently fewer babies in each group were examined at week 7, 11, and 15. The addition of iron to both casein and whey formulas discouraged colonisation and growth of staphylococci and bacteroids but encouraged the colonisation and growth of clostridia and enterococci. The type of protein and not the iron content had more effect upon the growth of bifidobacteria; both whey formulas, fortified or not, encouraged the colonisation by bifidobacteria. If an infant formula, for use in the first few months, is to mimic the physiological effects of breast milk, there may be microbiological arguments for not fortifying it with iron. However, large empirical trials would be necessary before advocating such a policy.     

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.     

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.     

Dietary vitamin E and polyunsaturated fatty acid (PUFA) in newborn babies with physiological jaundice

We studied 69 term babies aged 2–8 days who had physiological jaundice and who were fed formula A (Ostermilk Complete; vitamin E, 0.46 mg per 100 ml; polyunsaturated fatty acid (PUFA), ? 0.08 g per 100 ml), or formula B (Cow and Gate Premium; vitamin E, 1.0 mg per 100 ml; PUFA, ?0.55 g per 100 ml) or breast milk. Babies fed formula B, with the greatest vitamin E and PUFA content, had a significantly higher mean plasma vitamin E level compared with those fed formula A, even as early as the second and third day. Breast fed babies, 2–3 days old, had a lower mean plasma vitamin E level compared with formula B fed babies, thereafter vitamin E levels in breast fed babies rose. The RBCs of babies fed formula B and breast milk were significantly less susceptible to hydrogen peroxide (H₂O₂) haemolysis compared with the RBCs of those fed formula A. Reduced susceptibility to H₂O₂ haemolysis in formula B fed babies was observed in those as young as 2–3 days. Susceptibility to H₂O₂ haemolysis did not correlate with haemoglobin concentration, plasma bilirubin nor with the reticulocyte count in babies on different feeds.We conclude that in term newborn babies the vitamin E and PUFA contents of the milk feeds influence plasma vitamin E levels and susceptibility of RBCs to H₂O₂ haemolysis, but do not have an important bearing on the occurrence of physiological jaundice.     

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.     

Diet and faecal flora in the newborn: breast milk and infant formula.

This study examined the faecal flora on days 4, 14, and 28 of 17 breast fed babies and 26 bottle fed babies receiving a modern infant formula based on demineralized whey. Generally among breast fed babies bifidobacteria and staphylococci were the predominant organisms, whereas in the formula fed babies the predominant organisms were enterococci, coliforms, and clostridia. Despite the extensive modification of cows'' milk to make an infant formula resemble human breast milk, the results are very similar to those previously reported with unmodified cows'' milk baby feeds. The exact dietary factor responsible for these microbiological differences is unclear and in succeeding papers we have looked at the effects of protein quality, in particular the content of whey proteins, casein, and lactoferrin.     

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.     

Supplementation of an adapted formula with bovine lactoferrin. 2. Effects on serum iron, ferritin and zinc levels

Breast milk provides an excellent supply of most nutrients for newborn infants. Infant formulae should be nutritionally comparable to breast milk especially with regard to critical nutrients like iron and other trace elements. Infant formulae supplemented with various amounts of bovine lactoferrin were given to two groups of infants. These infants were compared with infants receiving unsupplemented formula and breast-fed infants. The effects of these diets on levels of haemoglobin, haematocrit, serum iron, ferritin and zinc were examined for a study period of 150 days. At birth, concentrations of iron, haemoglobin, haematocrit and zinc were comparable in all four feeding groups. The fact that the serum zinc level was not altered by lactoferrin supplementation appears to rule out an in-vivo effect of lactoferrin on zinc nutrition of infants. Ferritin levels of breast-fed infants were significantly higher than in non-supplemented formula-fed infants at day 30 and day 90. This difference was seen only at day 30, when comparing breast-fed infants to lactoferrin-supplemented formula-fed infants. Comparing the infants receiving formulae, the formula supplemented with the higher amount of bovine lactoferrin induced significantly higher serum ferritin levels compared to the unsupplemented formula at day 90 and day 150. These observations favour the idea that lactoferrin may be involved in iron absorption. Since this effect was pronounced only after 90 days, it has to be discussed as to whether this effect is a convincing argument for supplementing infant formulae with bovine lactoferrin.     

Current aspects of infant nutrition

It is still an important duty for pediatricians to inform parents about infant nutrition. An effort to insure successful breastfeeding in newborns is particularly necessary in order to avoid the introduction of foreign proteins, especially in high risk children. Allergy prevention is thus instigated early on. If necessary, a hypoallergenic milk may be used. Recently, there has been great concern that a high content of dioxine in breast milk exists, higher than in infant formulas. However, no evidence of toxicity has been noticed to date in breast fed children due to dioxine. Therefore, because of the many advantages, breast feeding should still be recommended for the first 4-6 months. In the last few years infant formulas have been adapted to simulate breastmilk by supplementation with taurine, carnitine and nucleotides. Most recently, Omega-3-fatty acids, which are important constituents of membrane phospholipids in the nervous system and the retina, have been added. In infant nutrition there is a trend nowadays toward unconventional forms of nutrition. An exclusive "lactoovo-vegetable" diet is able to meet all the requirements of a growing child. The critical components of a vegetarian diet are iron, calcium, vitamin B12 and vitamin D. These few examples demonstrate how important a nutrition-committee could be in elaborating basic information for the pediatrician, which would be useful in his daily work.     

Severe protracted diarrhea in infancy ("intractable diarrhea")

In most cases, protracted diarrhea in infants is due to lack of human breast milk, and iatrogenic malnutrition. Damage to the mucosa of the small intestine maintained by cow's milk proteins and malnutrition constitute main parts of a vicious circle interrupted by completely parenteral nutrition for some time, and slow reintroduction of human breast milk. This kind of therapy reduced mortality significantly. The best prophylaxis is the full support of breast feeding. As differential diagnosis the familial lethal diarrhea of the breast fed infant has to be considered. Furthermore, in case of diarrhea lasting for longer than two weeks, other less frequent causes have to be thought of, as pointed out briefly.