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Gastric luminal digestion of lactoferrin and transferrin by preterm infants
127
Early Human Development, 19 (1989) 127-135 Elsevier Scientific Publishers Ireland Ltd. EHD 00959
Gastric luminal digestion of lactoferrin and transferrin by preterm infants John. R. Britton and Otakar Koldovsk$ University of Arizona Health Sciences Center, Department of Pediatrics and Children’s Research Center, 1501 North CampbellAvenue, Tucson, AZ 85724, U.S.A. Accepted for publication 10 October 1988
Summary Lactoferrin, a milk iron-binding protein, may play antimicrobial, iron-absorptive and growth-promoting roles in the developing gastrointestinal tract. To perform such functions, lactoferrin must survive digestive processes in the gut lumen in an active form. We investigated the gastric digestion of lactoferrin in addition to that of the other milk proteins, transferrin and casein, in preterm infants by measuring their degradation during incubation in vitro at 37OC with gastric fluid at pH 1.8, 3.2 and 5.8. Fluid was obtained 1 h after a milk feeding, a time of maximum peptic activity, from 12 infants with a mean gestational age of 29.7 + 0.8 weeks at birth and a postnatal age of 24.7 f 3.2 days at sampling. Hydrolysis of all three proteins as indicated by generation of trichloroacetic acid soluble material from iodinated substrate was maximal at acid pH and declined by greater than 75% at pH 5.8, lactoferrin was less rapidly degraded than casein at low pH and transferrin breakdown was intermediate. Analysis of reaction mixtures by SDS-polyacrylamide gel electrophoresis showed degradation of lactoferrin and transferrin to low molecular weight products at pH 3.2 but minimal breakdown at pH 5.8. Several discrete fragments were generated at low pH, including species with molecular weights of 41,000-42,000 which may represent half-molecules. We conclude that dietary lactoferrin and transferrin may be degraded by preterm infant gastric fluid to discrete species, but that hydrolysis may be minimal at the prevailing postprandial pH. Consequently they may be rendered available for possible subsequent biological action within the infant. lactoferrin; transferrin;
casein; stomach; proteolysis.
Correspondence to: J.R. Britton. 0378-3782/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland Published and Printed in Ireland
Ltd.
128
Introduction Lactoferrin, the major iron-binding protein in human milk, has a high affinity for iron, a property which may be responsible for several of its proposed biological roles [l]. In vitro, lactoferrin inhibits the rowth of a variety of bacteria by sequestering iron in the medium required for their growth [ 1,2]. Consequently, it has been proposed that this protein may play a protective role by limiting the growth of potential pathogens within the gastrointestinal tract of the developing infant [I]. Lactoferrin receptors are present in the enteric mucosa of several species 13-51, and a role in the facilitation of mucosal iron absorption via receptor-mediated endocytosis has been considered [6,7]. In cell culture, lactoferrin may serve as a growth factor [8], and mitogenic effects upon the small intestinal epithelial cell have recently been demonstrated [9]. Milk feedings in experimental animals are trophic for the developing gastrointestinal tract [lo], and it is possible that lactoferrin might account for some of this effect. For lactoferrin to perform any of these functions within the developing gut, the protein must survive digestive processes within the gastrointestinal lumen in an active form. Substantial survival is suggested by the finding of lactoferrin in stool of breast-fed infants in which excretion is greater than that of formula-fed controls [ll-131. Moreover, lactoferrin excretion by the breast-fed infant declines with progressive postnatal age in a fashion similar to its decline in milk [ 141. However, excretion does not correlate with lactoferrin intake [12], suggesting that other factors such as endogenous production [12], might influence levels in the digesta. That gastrointestinal degradation of the protein also occurs is suggested by the observation that the percentage of ingested lactoferrin excreted decreases with postnatal age, perhaps due to the increasing capacity for protein digestion in early infancy [14]. In addition, fragments of lactoferrin have been detected in the stool by some [ 11,151 but not other [ 141workers, and such fragments are similar to those generated in vitro by purified pancreatic proteases. Thus although substantial gastrointestinal survival of lactoferrin appears likely, at least limited proteolysis probably also occurs. The sites of such proteolyis within the gut remain to be determined, however. In an initial approach to this problem, we have evaluated the gastric digestion of lactoferrin by meauring its hydrolysis by human stomach fluid, since this is one of the first digestive secretions to which the protein is exposed following ingestion. We also studied the in vitro gastric hydrolysis of transferrin, a related iron-binding protein present in human milk which may similarly function in iron uptake, bacteriostasis, and growth-promotion [2,6]. For comparison, the digestibility of bovine casein, a protein usually considered to be easily digestible, was studied. Because of the potential importance of lactoferrin and transferrin for the preterm infant, we utilized gastric fluid from a group of such infants following feeding-induced stimulation of gastric secretion. Methods Collection of gastricfluid This study was approved by the Human Subjects Committee of the University Of
129
Arizona Health Sciences Center, and informed parental consent was obtained prior to collection of samples. Twelve premature infants with a gestational age at birth of 29.7 f 0.8 (mean f S.E.M.) weeks were studied at a postnatal age of 24.7 & 3.2 days. All infants were receiving intermittent orogastric feedings of either Similac Special Care formula (9 infants) or human milk fortified with Natural Care (Ross Laboratories, Columbus, OH) (3 infants). The infants received a test feeding between 0800 and 1200 h on the morning of collection in the amount of 16.5 + 0.7 ml/kg body wt. Because maximum gastric luminal peptic activity has been reported to occur 60 min after a feeding in preterm infants [ 171, gastric fluid was collected at this time. The fluid was collected into plastic containers on ice, diluted 1 : 5 with normal saline, and homogenized with a glass-teflon homogenizer. It was subsequently assayed for proteolytic activity as described below. Dilution of the fluid was necessary because the large amount of undigested curd present at the time of collection rendered it difficult to pipette; in addition, dilution was also performed to obtain linearity of product generation with added fluid in the proteolytic assay described below. Analysis of protein hydrolysis The pH values of the gastric aspirates obtained were between 5.5 and 6, in agreement with early reports of the pH of gastric contents of infants 60 min after a feeding [18-211. Consequently, proteolysis was assayed at pH 5.8 in 0.1 M maleate buffer; in addition, measurements were performed at pH 1.8 (0.05 M HCl) and pH 3.2 (0.1 M glycine-HCl). These buffers are not known to interfere with gastric proteolytic activity [27,30]. The rate of hydrolysis was measured at 37OC in a 0.15 ml reaction mixture containing 0.05 ml of gastric fluid and 100 pg of the appropriate protein labelled with lz51[22]. Lactoferrin, human transferrin, and bovine alpha casein were obtained from Sigma; lactoferrin and transferrin had iron saturations of < 1% [23]. Following incubation for various times, the reaction was terminated by the addition of 1.0 ml of 10% trichloracetic acid (TCA) containing 0.2% bovine serum albumin and chilling on ice. The rate of hydrolysis was determined by measuring the generation of TCA-soluble radioactivity as previously described [24,25] under conditions of linearity with respect to time and amount of added gastric fluid. Such linearity was observed for all three proteins at each pH value tested. For each protein, hydrolysis was expressed as micrograms rendered TCA soluble per minute in the standard reaction mixture. Comparisons between rates of hydrolysis of different proteins and between rates of degradation of the same protein at different pH values were expressed as a percentage of the maximal activity observed. Electrophoretic analysis of reaction products In some experiments with lactoferrin and transferrin, reaction mixtures were prepared as described above but with gastric fluid pooled from 5 to 10 infants, and incubations were performed for various times at 37 OC. The reaction was terminated by additon of an equal volume of the sample buffer of Laemmli [26], followed by boiling for 5 min. Samples were subjected to electrophoresis in 10% polyacrylamide gels containing sodium dodecyl sulfate (SDS) as previously described [24]. Gels were stained with Coomassie blue and dried with heat under vacuum; autoradiography of
130
dried gels was performed at 25OC using Kodak RP-XOMAT calibrated with a molecular weight marker kit (Sigma).
film. Gels were
Analysis of data Data are expressed as the mean f standard error (S.E.M.) of determinations on each gastric fluid. Comparison between different groups was performed using ANOVA and Student’s t-test, with significance accepted at P< 0.05. Results The pH of the gastric contents rises above 5 postprandially in early infancy and remains high for greater than an hour [ 181. Accordingly, we evaluated the hydrolysis of lactoferrin, transferrin and casein at pH 5.8, in addition to pH 3.2 and 1.8. The relative rates of hydrolysis at each pH value are shown for the three proteins in Fig. 1. For casein and transferrin, maximal proteolysis was observed at pH 1.8, and the rate of hydrolysis fell at higher pH values such that less than 20% of the activity remained at pH 5.8. In contrast, lactoferrin degradation was maximal at pH 3.2 but fell to significantly lower values at both the lower and upper pH values tested. The rates of hydrolysis of the three proteins were subsequently compared at each pH value tested (Fig. 2). At pH 1.8, there were significant differences in the rates of degradation of all three proteins: casein was more rapidly degraded than lactoferrin, and transferrin was hydrolyzed at an intermediate rate. At pH 3.2 there was no significant difference in the proteolysis of casein and transferrin, but lactoferrin was degraded at a rate which was significantly less than that of the other two proteins. At pH 5.8 there were no significant differences in the rates of hydrolysis of any of the
Fig. 1. Gastric hydrolysis of milk proteins at various pH values. For casein (C), transferrin (T) and lactoferrin (L) the rates of hydrolysis at each pH value are expressed as a percentage of the maximal activity observed for the protein. Closed bars, pH 1.8; open bars, pH 3.2; hatched bars, pH 5.8. Maximum activities at respective pH optima expressed as micrograms hydrolyzed/minute are: C, 11.47 + 2.86; T, 8.12 * 2.06; L, 4.19 f 1.25 (mean f S.E.M., n = 12). All differences are significant except for lactoferrin at pH 1.8 and 5.8
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1.8
3.2 PH
5.8
Fig. 2. Relative rates of gastric hydrolysis of milk proteins at various pH values. For casein (closed bars), transferrin (open bars), and lactoferrin (hatched bars) the rates of hydrolysis at each pH shown are expressed as a percentage of the activity observed for the protein with the greatest rate of hydrolysis. The asterisk indicates a significant difference in relative hydrolysis compared to the other proteins at a given PH.
5.8 3.2
3.2 PH
PH Fig, 3. SDS polyacrylamide gel electrophoresis of reaction mixtures. Hydrolysis of lactoferrin (A) and transferrin (B) was performed at pH 5.8 and 3.2 for 60 min and reaction mixtures were analyzed by electrophoresis as described in Methods. Slot T, is the electrophoretic profile of a zero time sample. Direction of migration is from top to bottom. Numbers at right indicate molecular weight (X 10-l) of major degradation products. LF and TF indicate lactoferrin and transferrin bands, respectively.
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proteins. Degradation rates were very low for all three proteins, and for some individual fluid protein hydrolysis was undetectable at this pH. Reaction mixtures with lactoferrin and transferrin were analyzed by polyacrylamide gel electrophoresis in the presence of SDS and are shown in Fig. 3. At acid pH, there was degradation of the protein bands, generating breakdown products of the molecular weights indicated in Fig. 3. Several new bands appeared for each protein at pH 3.2, including species of approximate molecular weights 41 ,OOO-42,000. In contrast, loss of both lactoferrin and transferrin bands was negligible at pH 5.8, although some hydrolysis might have escaped detection by this method. Identical bands were obtained with both stained gels and autoradiograms. Discussion Our data suggest that lactoferrin and transferrin may be hydrolyzed in the stomach of the preterm infant, but that this process is pH dependent. During infancy, milk feeding results in an increase in gastric pH which may reach the range between 5 and 6.5 by 1 h after beginning a feeding [18-211. This neutralization of gastric acidity may be mediated by a number of milk components, including the buffering capacity of milk proteins. Although there is a gradual decline in gastric pH thereafter in most infants, some exhibit persistent elevations [20]. Such pH values are suboptimal for peptic activity [27], and as demonstrated by our data they result in diminished hydrolysis of the three proteins evaluated. Thus, despite the reportedly maximal acid proteolytic activity 1 h postprandially [ 171, the prevailing pH values in the stomach at that time favor protein survival. For lactoferrin and transferrin, proteins which may play biological roles in the gastrointestinal tract, such survival might enhance the potential for biological activity. An unexpected finding in our study was that although hydrolysis of casein and transferrin was optimal at pH 1.8, that of lactoferrin was greatest at pH 3.2, declining under more acidic conditions. Lactoferrin has a high isoelectric point [2], and precipitation might be expected at low pH values; however, none was observed under the conditions of our assay, and the reaction remained linear for its duration. Loss of lactoferrin iron, which is known to occur at low pH and to be associated with increased proteolytic susceptibility [2,28], is also an unlikely explanation since the lactoferrin utilized had a very low iron saturation, like that in milk [29]. Although the reason for this observation is unclear, it indicates that the pH range for gastric proteolytic susceptibility of lactoferrin is narrower than that of the other proteins, imposing a futher limitation on its digestibility. The relative digestibility of the three proteins at pH 1.8 was in the order casein > transferrin > lactoferrin. With increasing pH, these differences became less significant, since at pH 3.2 casein and transferrin showed similar rates of hydrolysis and at pH 5.8 no significant differences in proteolysis of the three proteins were observed. These observations illustrate that individual proteins may differ in their susceptibility to luminal gastric digestion, and that differences among proteins may be pH dependent.
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Like all assays utilizing proteins as substrates [30], the one employed in these studies has several limitations. Because the method measures the generation of TCA soluble material, no distinction is made between the production of large soluble peptides and free amino acids. Peptides which do not contain iodotyrosine will also escape detection. Moreover, although the proteins were labelled under conditions yielding at most one iodine per molecule, they differ in their tyrosine content and hence the distribution of iodination sites within the molecule. However, lactoferrin and transferrin share substantial sequence homology [2], making comparisons between these proteins less difficult. In addition, our experiments with polyacrylamide gel electrophoresis, which led to similar conclusions, circumvent many of the limitations of the proteolytic assay. Our results differ from those of Spik et al. [II], who found that lactoferrin was not hydrolyzed in vitro by the gastric secretions of infants during a 4-h incubation. The reasons for this difference are unclear, but several possible explanations exist. These workers did not specify the pH of their incubation mixture or whether the gastric fluid was obtained from fed or fasted infants, factors which could influence fluid proteolytic activity. Differences may have been due to the age ranges of study subjects: they evaluated fluid from I-3-month-old term infants whereas we studied preterm infants at a mean age of approximately 30 days. Finally, partially and completely iron-saturated lactoferrin were utilized, in contrast to apolactoferrin as in our experiments. We have employed apolactoferrin becuase of the reportedly low iron saturation of this protein in human milk [29]; unsaturated lactoferrin is known to have greater proteolytic susceptibility than the saturated form [2,28]. These factors may account for at least part of the differences in the two studies. The electrophoretic experiments indicate that several lower molecular weight digestion products of similar molecular weights may result from gastric digestion of both lactoferrin and transferrin. The major degradation products of lactoferrin and transferrin had molecular weights of 42,000 and 41,000, respectively. These species may represent half molecules of the native monomers, each of which has a molecular weight of approximately 79,000 [2]. Iron saturated lactoferrin is cleaved in vitro by pepsin at pH 3 .O into fragments of approximately 33,000 molecular weight which probably represent the C-terminal half of the molecule [31]. Consistent with these reports, we observed a lactoferrin degradation product of 34,000, in addition to the 42,000 species and several other breakdown products. Of note is that Spik et al. [l l] found peptides of molecular weights 34,000 and 42,000 in immunologically detectable preparations of lactoferrin from the stool of infants fed human milk. More recently, Goldman et al. [15] described lactoferrin fragments of 39-43, 35-38 and 30-34 kDa in the stool of premature infants fed fortified human milk. Although these workers noted that the fragments were similar to those produced in vitro by treating apolactoferrin with trypsin or chymotrypsin, our finding that gastric fluid from preterm infants may produce species of similar size suggests that some might originate in the stomach. In fact, the peptic, tryptic and chymotryptic fragments of iron-saturated lactoferrin are similar [32]; whether this is also true for apolactoferrin is unknown. Two stool lactoferrin fragments detected in vivo by Spik et al. were able
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to bind iron [l 11,and it is possible that some of the fragments observed in our own and other [32] studies may retain biological activity. Further purification of fragments generated at different levels of the gastrointestinal tract and evaluation of their functional activity will be necessary to address this question. Acknowledgements The authors thank M. Morrill for expert technical assistance. This study was supported by grant number AM27624 from the National Institutes of Health, and grants from the Nestle Research Foundation and Flinn Foundation. References 1
Brock, J.H. (1980): Lactoferrin in human milk: its role in iron absorption and protection against enteric infection in the newborn infant. Arch. Dis. Child., 55,417-421. 2 Brock, J.H. (1985): Transferrins. In: Metallo-proteins, pp. 183-262. Editor: P.M. Harrison. Verlag Chemie, Weinheim. 3 Cox, T.M., Mazurier, J., Spik, G., Montreuil, J. and Peters, T.J. (1979): Iron-binding proteins and influx of iron across the duodenal brush border. Evidence for specific lactotransferrin receptors in the human intestine. Biochim. Biophys. Acta., 588,120-128. Davidson, L.A. and Lonnerdal, B. (1988): Specific binding of lactoferrin to brush-border membrane: ontogeny and effect of glycan chain. Am. J. Physiol., 254, G580-G585. Hu, W-L., Mazurier, J., Sawatzki, G., Montreul, J. and Spik, G. (1988): Lactoferrin receptor of mouse small-intestinal brush border. Biochem. J., 249,435-441. Huebers, H.A., Hueber, E., Csiba. E., Rummell, W. and Finch, C.A. (1983): The significance of transferrin for intestinal iron absorption. Blood, 61,283-290. Fair-weather-Tait, S.J., Balmer, S.E., Scott, P.H. and Minski, M.J. (1987): Lactoferrin and iron absorption in newborn infants. Pediatr. Res., 22.651-654. Hashizume, S., Kuroda, K. and Murakami, H. (1983): Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium. Biochim. Biophys. Acta., 763,377-382. 9 Nichols, B.L., McKee, K.S., Henry, J.F. and Putman, M. (1987): Human lactoferrin stimulates thymidine incorporation into DNA of rat crypt cells. Pediatr. Res., 21,563-567. 10 Koldovsky, 0. and Thornburg, W. (1987): Hormones in milk. J. Pediatr. Gastroenterology Nutr., 6,172-l%. 11 Spik, G., Brunet, B., Mazurier-Dehaine. C., Fontaine, G. and Montreuil, J. (1982): Characterization and properties of human and bovine lactotransferrins extracted from the faeces of newborn infants. Acta. Paediatr. Stand., 71,979-985. 12 Shanler, R.J., Goldblum, R.M., Garza, C. and Goldman, A.S. Enhanced fecal excretion of selected immune factors in very low birth weight infants fed fortified human milk. Pediatr. Res., 20, 711715. 13 Prentice, A., Ewing, G., Roberts, S.B., Lucas, A., MacCarthy, A., Jarjou, L.M.A. and Whitehead, R.G. (1988): The nutritional role of breast-milk IgA and lactoferrin. Acta. Paediatr. Stand., 76,592-598. Davidson, L.A. and Lonnerdal, B. (1988): Persistence of human milk proteins in the breast-fed infant. Acta, Paediatr. Stand., 76,733-740. Goldman, A.S., Garza, C., Goldblum, R.M. and Schanler, R.J. (1988): Molecular forms of lactoferrin in stools and urine from infants fed human milk. Pediatr. Res., 23,304A. Huebers, H.A. and Finch, C.A. (1987): The physiology of transferrin and transferrin receptors. Physiol. Rev., 67,520-582. Yahav, J., Carrion, V., Lee, P.C. and Lebenthal, E. (1987): Meal-stimulated pepsinogen secretion in premature infants. J. Pediatr., 110,949-951.
135 18 19 20
21 22 23 24 25 26 27 28
29
30 31 32
Mason, S. (1962): Some aspects of gastric function in the newborn. Arch. Dis. Child., 37,387-391. Harries, J.T., and Fraser, A.J. (1968): The acidity of the gastric contents of premature babies during the first fourteen days of life. Biol. Neonate, 12,186-193. Hodge, C., Lebenthal, E., Lee, P.C. and Topper, W. (1983): Amylase in the saliva and in the gastric aspirates of premature infants: its potential role in glucose polymer hydrolysis. Pediatr. Res., 17, 998-1001. Cavell, B. (1983): Postprandial gastric acid secretion in infants. Acta. Paediatr. &and., 72, 857860. Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1963): The preparation of lUI-labelled human growth hormone of high specific radioactivity. Biochem. J., 89, 114-123. Stookey, L.L. (1970): Ferrozine - a new spectrophotometric reagent for iron. Anal. Chem., 42, 719-181. Britton, J.R. and Koldovsky, 0. (1987): Luminal digestion of lactoferrin in suckling and weanling rats. Am. J. Physiol., 253, G397-G403. Britton, J.R. and Koldovsky, 0. (1988): Development of luminal protein digestion in suckling and weanling rats. Biol. Neonate, 53,39-46. Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophase T,. Nature (London), 227.680-682. Defize, J. and Meuwissen. S.G.M. (1987): Pepsinogens: an update of biochemical, physiological, and clinical aspects. J. Pediatr. Gastroenterol. Nutr., 6,493-508. Brines, R.D. and Brock, J.H. (1983): The effect of trypsin and chymotrypsin on the in vitro antimicrobial and iron-binding properties of lactoferrin in human milk and bovine colostrum. Unusual resistance of human apolactoferrin to proteolytic digestion. Biochim. Biophys. Acta, 759, 229235. Fransson, G.-B. and Liinnerdal, B. (1980): Iron in human milk. J. Pediatr., 96,380-384. Bergmeyer, H.U. (1983): Methods of Enzymatic Analysis, Vol. 5, pp. 99-155. Verlag Chemie, Weinheim. Line, W.F., Sly, D.A. and Bezkorovainy, A. (1976): Limited cleavage of human lactoferrin with pepsin. Int. J. B&hem., 7,203-208. Bluard-Deconinck, J.-M., Williams, J., Evans, R.W., Van Snick, J., Osinski, P.A. and Masson, P.L. (1978): Iron-binding fragments from the N-terminal and C-terminal regions of human lactoferrin. Biochem. J., 171,321-327.
Early Human Development, 19 (1989) 127-135 Elsevier Scientific Publishers Ireland Ltd. EHD 00959
Gastric luminal digestion of lactoferrin and transferrin by preterm infants John. R. Britton and Otakar Koldovsk$ University of Arizona Health Sciences Center, Department of Pediatrics and Children’s Research Center, 1501 North CampbellAvenue, Tucson, AZ 85724, U.S.A. Accepted for publication 10 October 1988
Summary Lactoferrin, a milk iron-binding protein, may play antimicrobial, iron-absorptive and growth-promoting roles in the developing gastrointestinal tract. To perform such functions, lactoferrin must survive digestive processes in the gut lumen in an active form. We investigated the gastric digestion of lactoferrin in addition to that of the other milk proteins, transferrin and casein, in preterm infants by measuring their degradation during incubation in vitro at 37OC with gastric fluid at pH 1.8, 3.2 and 5.8. Fluid was obtained 1 h after a milk feeding, a time of maximum peptic activity, from 12 infants with a mean gestational age of 29.7 + 0.8 weeks at birth and a postnatal age of 24.7 f 3.2 days at sampling. Hydrolysis of all three proteins as indicated by generation of trichloroacetic acid soluble material from iodinated substrate was maximal at acid pH and declined by greater than 75% at pH 5.8, lactoferrin was less rapidly degraded than casein at low pH and transferrin breakdown was intermediate. Analysis of reaction mixtures by SDS-polyacrylamide gel electrophoresis showed degradation of lactoferrin and transferrin to low molecular weight products at pH 3.2 but minimal breakdown at pH 5.8. Several discrete fragments were generated at low pH, including species with molecular weights of 41,000-42,000 which may represent half-molecules. We conclude that dietary lactoferrin and transferrin may be degraded by preterm infant gastric fluid to discrete species, but that hydrolysis may be minimal at the prevailing postprandial pH. Consequently they may be rendered available for possible subsequent biological action within the infant. lactoferrin; transferrin;
casein; stomach; proteolysis.
Correspondence to: J.R. Britton. 0378-3782/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland Published and Printed in Ireland
Ltd.
128
Introduction Lactoferrin, the major iron-binding protein in human milk, has a high affinity for iron, a property which may be responsible for several of its proposed biological roles [l]. In vitro, lactoferrin inhibits the rowth of a variety of bacteria by sequestering iron in the medium required for their growth [ 1,2]. Consequently, it has been proposed that this protein may play a protective role by limiting the growth of potential pathogens within the gastrointestinal tract of the developing infant [I]. Lactoferrin receptors are present in the enteric mucosa of several species 13-51, and a role in the facilitation of mucosal iron absorption via receptor-mediated endocytosis has been considered [6,7]. In cell culture, lactoferrin may serve as a growth factor [8], and mitogenic effects upon the small intestinal epithelial cell have recently been demonstrated [9]. Milk feedings in experimental animals are trophic for the developing gastrointestinal tract [lo], and it is possible that lactoferrin might account for some of this effect. For lactoferrin to perform any of these functions within the developing gut, the protein must survive digestive processes within the gastrointestinal lumen in an active form. Substantial survival is suggested by the finding of lactoferrin in stool of breast-fed infants in which excretion is greater than that of formula-fed controls [ll-131. Moreover, lactoferrin excretion by the breast-fed infant declines with progressive postnatal age in a fashion similar to its decline in milk [ 141. However, excretion does not correlate with lactoferrin intake [12], suggesting that other factors such as endogenous production [12], might influence levels in the digesta. That gastrointestinal degradation of the protein also occurs is suggested by the observation that the percentage of ingested lactoferrin excreted decreases with postnatal age, perhaps due to the increasing capacity for protein digestion in early infancy [14]. In addition, fragments of lactoferrin have been detected in the stool by some [ 11,151 but not other [ 141workers, and such fragments are similar to those generated in vitro by purified pancreatic proteases. Thus although substantial gastrointestinal survival of lactoferrin appears likely, at least limited proteolysis probably also occurs. The sites of such proteolyis within the gut remain to be determined, however. In an initial approach to this problem, we have evaluated the gastric digestion of lactoferrin by meauring its hydrolysis by human stomach fluid, since this is one of the first digestive secretions to which the protein is exposed following ingestion. We also studied the in vitro gastric hydrolysis of transferrin, a related iron-binding protein present in human milk which may similarly function in iron uptake, bacteriostasis, and growth-promotion [2,6]. For comparison, the digestibility of bovine casein, a protein usually considered to be easily digestible, was studied. Because of the potential importance of lactoferrin and transferrin for the preterm infant, we utilized gastric fluid from a group of such infants following feeding-induced stimulation of gastric secretion. Methods Collection of gastricfluid This study was approved by the Human Subjects Committee of the University Of
129
Arizona Health Sciences Center, and informed parental consent was obtained prior to collection of samples. Twelve premature infants with a gestational age at birth of 29.7 f 0.8 (mean f S.E.M.) weeks were studied at a postnatal age of 24.7 & 3.2 days. All infants were receiving intermittent orogastric feedings of either Similac Special Care formula (9 infants) or human milk fortified with Natural Care (Ross Laboratories, Columbus, OH) (3 infants). The infants received a test feeding between 0800 and 1200 h on the morning of collection in the amount of 16.5 + 0.7 ml/kg body wt. Because maximum gastric luminal peptic activity has been reported to occur 60 min after a feeding in preterm infants [ 171, gastric fluid was collected at this time. The fluid was collected into plastic containers on ice, diluted 1 : 5 with normal saline, and homogenized with a glass-teflon homogenizer. It was subsequently assayed for proteolytic activity as described below. Dilution of the fluid was necessary because the large amount of undigested curd present at the time of collection rendered it difficult to pipette; in addition, dilution was also performed to obtain linearity of product generation with added fluid in the proteolytic assay described below. Analysis of protein hydrolysis The pH values of the gastric aspirates obtained were between 5.5 and 6, in agreement with early reports of the pH of gastric contents of infants 60 min after a feeding [18-211. Consequently, proteolysis was assayed at pH 5.8 in 0.1 M maleate buffer; in addition, measurements were performed at pH 1.8 (0.05 M HCl) and pH 3.2 (0.1 M glycine-HCl). These buffers are not known to interfere with gastric proteolytic activity [27,30]. The rate of hydrolysis was measured at 37OC in a 0.15 ml reaction mixture containing 0.05 ml of gastric fluid and 100 pg of the appropriate protein labelled with lz51[22]. Lactoferrin, human transferrin, and bovine alpha casein were obtained from Sigma; lactoferrin and transferrin had iron saturations of < 1% [23]. Following incubation for various times, the reaction was terminated by the addition of 1.0 ml of 10% trichloracetic acid (TCA) containing 0.2% bovine serum albumin and chilling on ice. The rate of hydrolysis was determined by measuring the generation of TCA-soluble radioactivity as previously described [24,25] under conditions of linearity with respect to time and amount of added gastric fluid. Such linearity was observed for all three proteins at each pH value tested. For each protein, hydrolysis was expressed as micrograms rendered TCA soluble per minute in the standard reaction mixture. Comparisons between rates of hydrolysis of different proteins and between rates of degradation of the same protein at different pH values were expressed as a percentage of the maximal activity observed. Electrophoretic analysis of reaction products In some experiments with lactoferrin and transferrin, reaction mixtures were prepared as described above but with gastric fluid pooled from 5 to 10 infants, and incubations were performed for various times at 37 OC. The reaction was terminated by additon of an equal volume of the sample buffer of Laemmli [26], followed by boiling for 5 min. Samples were subjected to electrophoresis in 10% polyacrylamide gels containing sodium dodecyl sulfate (SDS) as previously described [24]. Gels were stained with Coomassie blue and dried with heat under vacuum; autoradiography of
130
dried gels was performed at 25OC using Kodak RP-XOMAT calibrated with a molecular weight marker kit (Sigma).
film. Gels were
Analysis of data Data are expressed as the mean f standard error (S.E.M.) of determinations on each gastric fluid. Comparison between different groups was performed using ANOVA and Student’s t-test, with significance accepted at P< 0.05. Results The pH of the gastric contents rises above 5 postprandially in early infancy and remains high for greater than an hour [ 181. Accordingly, we evaluated the hydrolysis of lactoferrin, transferrin and casein at pH 5.8, in addition to pH 3.2 and 1.8. The relative rates of hydrolysis at each pH value are shown for the three proteins in Fig. 1. For casein and transferrin, maximal proteolysis was observed at pH 1.8, and the rate of hydrolysis fell at higher pH values such that less than 20% of the activity remained at pH 5.8. In contrast, lactoferrin degradation was maximal at pH 3.2 but fell to significantly lower values at both the lower and upper pH values tested. The rates of hydrolysis of the three proteins were subsequently compared at each pH value tested (Fig. 2). At pH 1.8, there were significant differences in the rates of degradation of all three proteins: casein was more rapidly degraded than lactoferrin, and transferrin was hydrolyzed at an intermediate rate. At pH 3.2 there was no significant difference in the proteolysis of casein and transferrin, but lactoferrin was degraded at a rate which was significantly less than that of the other two proteins. At pH 5.8 there were no significant differences in the rates of hydrolysis of any of the
Fig. 1. Gastric hydrolysis of milk proteins at various pH values. For casein (C), transferrin (T) and lactoferrin (L) the rates of hydrolysis at each pH value are expressed as a percentage of the maximal activity observed for the protein. Closed bars, pH 1.8; open bars, pH 3.2; hatched bars, pH 5.8. Maximum activities at respective pH optima expressed as micrograms hydrolyzed/minute are: C, 11.47 + 2.86; T, 8.12 * 2.06; L, 4.19 f 1.25 (mean f S.E.M., n = 12). All differences are significant except for lactoferrin at pH 1.8 and 5.8
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1.8
3.2 PH
5.8
Fig. 2. Relative rates of gastric hydrolysis of milk proteins at various pH values. For casein (closed bars), transferrin (open bars), and lactoferrin (hatched bars) the rates of hydrolysis at each pH shown are expressed as a percentage of the activity observed for the protein with the greatest rate of hydrolysis. The asterisk indicates a significant difference in relative hydrolysis compared to the other proteins at a given PH.
5.8 3.2
3.2 PH
PH Fig, 3. SDS polyacrylamide gel electrophoresis of reaction mixtures. Hydrolysis of lactoferrin (A) and transferrin (B) was performed at pH 5.8 and 3.2 for 60 min and reaction mixtures were analyzed by electrophoresis as described in Methods. Slot T, is the electrophoretic profile of a zero time sample. Direction of migration is from top to bottom. Numbers at right indicate molecular weight (X 10-l) of major degradation products. LF and TF indicate lactoferrin and transferrin bands, respectively.
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proteins. Degradation rates were very low for all three proteins, and for some individual fluid protein hydrolysis was undetectable at this pH. Reaction mixtures with lactoferrin and transferrin were analyzed by polyacrylamide gel electrophoresis in the presence of SDS and are shown in Fig. 3. At acid pH, there was degradation of the protein bands, generating breakdown products of the molecular weights indicated in Fig. 3. Several new bands appeared for each protein at pH 3.2, including species of approximate molecular weights 41 ,OOO-42,000. In contrast, loss of both lactoferrin and transferrin bands was negligible at pH 5.8, although some hydrolysis might have escaped detection by this method. Identical bands were obtained with both stained gels and autoradiograms. Discussion Our data suggest that lactoferrin and transferrin may be hydrolyzed in the stomach of the preterm infant, but that this process is pH dependent. During infancy, milk feeding results in an increase in gastric pH which may reach the range between 5 and 6.5 by 1 h after beginning a feeding [18-211. This neutralization of gastric acidity may be mediated by a number of milk components, including the buffering capacity of milk proteins. Although there is a gradual decline in gastric pH thereafter in most infants, some exhibit persistent elevations [20]. Such pH values are suboptimal for peptic activity [27], and as demonstrated by our data they result in diminished hydrolysis of the three proteins evaluated. Thus, despite the reportedly maximal acid proteolytic activity 1 h postprandially [ 171, the prevailing pH values in the stomach at that time favor protein survival. For lactoferrin and transferrin, proteins which may play biological roles in the gastrointestinal tract, such survival might enhance the potential for biological activity. An unexpected finding in our study was that although hydrolysis of casein and transferrin was optimal at pH 1.8, that of lactoferrin was greatest at pH 3.2, declining under more acidic conditions. Lactoferrin has a high isoelectric point [2], and precipitation might be expected at low pH values; however, none was observed under the conditions of our assay, and the reaction remained linear for its duration. Loss of lactoferrin iron, which is known to occur at low pH and to be associated with increased proteolytic susceptibility [2,28], is also an unlikely explanation since the lactoferrin utilized had a very low iron saturation, like that in milk [29]. Although the reason for this observation is unclear, it indicates that the pH range for gastric proteolytic susceptibility of lactoferrin is narrower than that of the other proteins, imposing a futher limitation on its digestibility. The relative digestibility of the three proteins at pH 1.8 was in the order casein > transferrin > lactoferrin. With increasing pH, these differences became less significant, since at pH 3.2 casein and transferrin showed similar rates of hydrolysis and at pH 5.8 no significant differences in proteolysis of the three proteins were observed. These observations illustrate that individual proteins may differ in their susceptibility to luminal gastric digestion, and that differences among proteins may be pH dependent.
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Like all assays utilizing proteins as substrates [30], the one employed in these studies has several limitations. Because the method measures the generation of TCA soluble material, no distinction is made between the production of large soluble peptides and free amino acids. Peptides which do not contain iodotyrosine will also escape detection. Moreover, although the proteins were labelled under conditions yielding at most one iodine per molecule, they differ in their tyrosine content and hence the distribution of iodination sites within the molecule. However, lactoferrin and transferrin share substantial sequence homology [2], making comparisons between these proteins less difficult. In addition, our experiments with polyacrylamide gel electrophoresis, which led to similar conclusions, circumvent many of the limitations of the proteolytic assay. Our results differ from those of Spik et al. [II], who found that lactoferrin was not hydrolyzed in vitro by the gastric secretions of infants during a 4-h incubation. The reasons for this difference are unclear, but several possible explanations exist. These workers did not specify the pH of their incubation mixture or whether the gastric fluid was obtained from fed or fasted infants, factors which could influence fluid proteolytic activity. Differences may have been due to the age ranges of study subjects: they evaluated fluid from I-3-month-old term infants whereas we studied preterm infants at a mean age of approximately 30 days. Finally, partially and completely iron-saturated lactoferrin were utilized, in contrast to apolactoferrin as in our experiments. We have employed apolactoferrin becuase of the reportedly low iron saturation of this protein in human milk [29]; unsaturated lactoferrin is known to have greater proteolytic susceptibility than the saturated form [2,28]. These factors may account for at least part of the differences in the two studies. The electrophoretic experiments indicate that several lower molecular weight digestion products of similar molecular weights may result from gastric digestion of both lactoferrin and transferrin. The major degradation products of lactoferrin and transferrin had molecular weights of 42,000 and 41,000, respectively. These species may represent half molecules of the native monomers, each of which has a molecular weight of approximately 79,000 [2]. Iron saturated lactoferrin is cleaved in vitro by pepsin at pH 3 .O into fragments of approximately 33,000 molecular weight which probably represent the C-terminal half of the molecule [31]. Consistent with these reports, we observed a lactoferrin degradation product of 34,000, in addition to the 42,000 species and several other breakdown products. Of note is that Spik et al. [l l] found peptides of molecular weights 34,000 and 42,000 in immunologically detectable preparations of lactoferrin from the stool of infants fed human milk. More recently, Goldman et al. [15] described lactoferrin fragments of 39-43, 35-38 and 30-34 kDa in the stool of premature infants fed fortified human milk. Although these workers noted that the fragments were similar to those produced in vitro by treating apolactoferrin with trypsin or chymotrypsin, our finding that gastric fluid from preterm infants may produce species of similar size suggests that some might originate in the stomach. In fact, the peptic, tryptic and chymotryptic fragments of iron-saturated lactoferrin are similar [32]; whether this is also true for apolactoferrin is unknown. Two stool lactoferrin fragments detected in vivo by Spik et al. were able
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to bind iron [l 11,and it is possible that some of the fragments observed in our own and other [32] studies may retain biological activity. Further purification of fragments generated at different levels of the gastrointestinal tract and evaluation of their functional activity will be necessary to address this question. Acknowledgements The authors thank M. Morrill for expert technical assistance. This study was supported by grant number AM27624 from the National Institutes of Health, and grants from the Nestle Research Foundation and Flinn Foundation. References 1
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