Biologically Active Polypeptides in Milk JOHN R. BRITTON, MD, PHD, ABBA J. KASTIN, MD

ABSTRACT: Many biologically active polypeptides have been described in the milk of several species. Various functions for these polypeptides in addition to nutrition have been proposed in the maternal body and in the breast-fed infant. These polypeptides are derived from several sources and multiple factors control their secretion into milk as well as their fate in the mother and infant. An increasing body of evidence supports the concept that they may function physiologically. KEY INDEXING TERMS: polypeptides; milk; breast feeding; milk proteins; hormones; growth factors; opiates. [Am J Med Sci 1991; 301(2):124-132.]

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he concept that proteins in milk may serve some physiological function other than providing digestive substrate is a relatively recent one. Milk from several species contains a diversity of proteins possessing biological activity, including enzymes, anti-infectious substances, and allergins,1-3 and various roles have been proposed for them in the body. In addition to the larger proteins, there exist in milk a variety of biologically active smaller proteins we shall term polypeptides hormones, in this review. These include substances1-5 commonly classified as hormones, growth factors, and opiates, such as prolactin, luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), growth hormone releasing hormone (GHRH), calcitonin, parathyroid hormonelike protein, erythropoietin, insulin, neurotensin, vasoactive intestinal peptide (VIP), oxytocin, relaxin, somatostatin, bombesin, delta-sleep-inducing peptide (DSIP), epidermal growth factor (EGF), nerve growth factor (NGF), transforming growth factors (TGF) alpha and beta, insulin-like growth factors (IGF) I and II, several mammary derived growth factors (MDGF) and a mammary derived growth inhibitor (MDGI), and the casomorphins and other casein-derived peptides such as morphiceptin, some with immunological activities.

From the Veterans Administration Medical Center and Tulane University School of Medicine, New Orleans, Louisiana. Address correspondence to: John R. Britton, MD, PhD, University of Utah Medical Center, 3878 East Adonis Drive, Salt Lake City, Utah 84124, (801)350·4654 Reprints not available.

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Many of these substances are present in milk in concentrations that exceed those in maternal blood and infant tissues. Such observations have led to speculation that milk might serve as a major source of these factors, either in the mother or the recipient infant. That milk might provide biologically active polypeptides for the infant is an attractive concept, since many such factors may otherwise be diminished in the newborn because of developmental delays in their synthesis. It is the purpose of this review to discuss biologically active polypeptides in milk with particular attention to three aspects: (a) the source of milk polypeptides and the factors influencing their concentrations; (b) the fate of milk polypeptides in the infant and mother; and (c) evidence that milk polypeptides may function physiologically other than by providing digestive substrate. The review will focus on the smaller polypeptides present in milk, although occasionally results regarding specific milk proteins will be discussed for illustrative purposes. Emphasis will be upon substances in the milk of humans, although information from experiments in animals will be included. Excellent recent reviews are available regarding related subjects, to which the interested reader is referred.1- 7 Sources of Milk Peptldes

The major milk proteins, such as the caseins and lactalbumin, are produced in mammary epithelial cells from their constituent amino acids. 6 After synthesis on the rough endoplasmic reticulum, they are modified by various processes (removal of signal peptides, phosphorylation, glycosylation) and concentrated into vesicles on the golgi apparatus, after which they are secreted into the alveolar epithelium by an exocytotic process.6 With the exception of those peptides derived by proteolytic digestion from the major milk proteins, little is known about the sources of biologically active polypeptides that appear in milk. Conceivably, such polypeptides might be synthesized in the mammary gland in a manner similar to that of the major milk proteins or be taken up from the plasma by trans- or paracellular processes and concentrated during secretion of the milk. Unfortunately, limited information is available regarding these possibilities, and the potential influence of factors such as peptide size, charge, and amino acid composition upon such processes remains almost completely unexplored. At least one protein in milk, transferrin, may originate by both mechanisms. This protein is not only February 1991 Volume 301 Number 2

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synthesized by the mammary gland of both the mouse and rabbit,S,9 but there is also evidence for its transfer from serum to milk in the mouse. 10 Whether or not milk polypeptides may behave similarly is unknown. It would seem reasonable that peptides with known extramammary sources might be transferred from plasma into milk and that changes in plasma concentrations of the peptides might result in corresponding changes in their milk content. This seems to be the case with several hypothalamic and pituitary peptides. Infusion of prolactin, GHRH, and TRH into the circulation of lactating rats results in an increase in their respective concentrations in milk.11-16 Injected prolactin is concentrated into milk in sheep,12 and bovine prolactin administered to lactating rats behaves similarly. Uptake is thought to be mediated by receptors located on the basal side of the alveolar secretory cells. Another important milk polypeptide that may originate from extra-mammary sources is EGF. Attempts to localize EGF in the mouse mammary gland by immunocytochemical methods have been unsuccessful17 and low amounts ofpre-pro-EGF messenger RNA have been detected in this organ. 1S This suggests that EGF is not synthesized there but transported across the mammary epithelium into milk. Uptake of EGF from the circulation is supported by observations in the lactating goat, in which intra-arterial infusion of radioactively-labeled EGF was followed by mammary uptake and subsequent appearance of the peptide in milk.19 Similar results were reported in mice. 20 In one study of human milk,21 EGF content closely correlated with the volume of milk expressed, suggesting passive transport by a transcellular process. Reports implicating the salivary glands as sources of milk EGF in mice are conflicting.2o,22,23 Caution must be exercised, however, in assuming that all milk polypeptides with known extramammary sources are simply transferred from plasma into milk. Evidence was recently presented that in addition to its known ovarian, uterine, and placental sources, relaxin may be synthesized by the guinea pig mammary gland. 24 The 'peptide was detected in mammary epithelia by immunocytochemistry with heterologous antisera during late pregnancy and lactation. Moreover, relaxin Cpeptide but not B-peptide probes hybridized with poly (A)+ RNA extracted from mammary tissue on day 6 of lactation. Since porcine probes were used, it is possible that species differences in nucleic acid sequence homologies of the Band C chains could account for this discrepancy. Nevertheless, these results suggest that the mammary gland may be a source of relaxin in the guinea pig. The mammary gland may also be a source for the parathyroid hormone-like protein recently reported in milk. 25,26 Multiple Forms of Milk Polypeptides

Several biologically active milk polypeptides have been shown to exist in multiple chromatographically THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

distinct forms. Multiple forms of LHRH, DSIP, calcitonin, neurotensin, EGF- and bombesin-like material have been demonstrated in milk,27-31 although detailed structural and functional characterization in each case has not been performed. At least two peptides, calcitonin and DSIP, have been detected in apparently large molecular weight forms in human milk as determined by gel filtation chromatography.28,29 Digestion in each case with trypsin yielded multiple species of apparently lower molecular weight. These large forms might represent true precursors or merely association of the peptides with larger molecular weight trypsin-sensitive material. Factors Influencing Milk Peptide Content

Although the bulk of milk protein has traditionally been considered a single variable, it has become increasingly apparent that different polypeptides, like other nutrients in milk, may vary considerably in their concentrations. Since the intake of a biologically active peptide by the infant may depend not only upon the amount of milk consumed but also upon the concentration of the peptide in milk, the determination of factors influencing such concentrations may be important in establishing the importance of milk as a soUrce of the peptide for the infant. For a given peptide, numerous factors could influence milk content, including circadian rhythms, number of infants suckled, hormones, stage and duration of lactation, post-conceptual timing, and mammary gland sampled. Unfortunately, information is limited regarding the relative importance of such factors, and no single peptide has been systematically studied with respect to all. Moreover, the relationship between such changes and changes in possible biological action within the recipient infant remain to be explored. Circadian Rhythms. The possibility of circadian variations in the concentrations of polypeptides in milk has received limited investigation. Concentrations of DSIP in human milk were noted to show a peak in the afternoon with a trough in the morning. 2s Possibly, the increased concentrations of this peptide in the afternoon might result in prolonged sleeping periods during the following night. In contrast, no circadian variation has been found for prolactin in human or bovine milk32,33 or EGF in human milk. 21 Number of Infants Suckled. The number of infants suckled could also be a factor in determination of the concentrations of a polypeptide in milk, but information on this aspect is available primarily in animals. In the lactating rat, concentrations of prolactin increased in milk as the number of pups suckled increased from 4 to 12.13 A similar phenomenon has been observed in pigs.34 Since pituitary secretion of rat prolactin is diminished in rats suckling smalllitters,35 low concentrations in milk may simply reflect low concentrations in serum. For transferrin in rat milk,36 no significant differences were observed between dams suckling 2 and 10 pups per litter when suckled glands were

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compared, although concentrations of this protein tended to be highest from nonsuckled glands of those dams with smaller litters. In women, similar patterns of change in insulin, growth hormone, and TSH were noted during the first 11 days postpartum in the milk of those who breast fed and those who did not,37 suggesting that for these hormones suckling did not influence the developmental patterns of milk concentration. Hormones. Although a complex interplay of several hormones may regulate mammary production of protein,7 little is known about the humoral factors influencing secretion of biologically-active polypeptides into milk. The secretion of those peptides derived from the major milk proteins such as the casomorphins (see 'below) might be expected to be controlled by the same processes regulating the production of the ' proteins from which they are derived. Since casein production by mammary epithelium is controlled by insulin, prolactin, and hydrocortisone,7 it is reasonable to speculate that the yield of casomorphins derived from casein should be similarly controlled. Limited results suggest hormonal regulation of the milk content of other peptides. For example, TRH administered to lactating goats increased the prolactin content of their milk as it did in blood;38 a similar effect was exerted in cows with the steroids dexamethasone and estradiol.39 Thyroid hormones may regulate the concentration of TSH in milk, although this may also be mediated by changes of the concentration of TSH in blood with subsequent changes in transport into milk; T3 administration to lactating rats decreased the concentrations of TSH in serum and milk simultaneously.40 Thyroidectomy led to an increase in concentrations of TSH in these fluids. 40 Other examples possibly related to endocrine factors include the elevation of concentrations of prolactin in milk from women with galactorrhea41 and the increased concentration of insulin in milk from obese women. 42 Duration of Milking. Unlike the detailed results regarding milk lipids,43 very little information is available regarding the pattern of secretion of polypeptides into milk during repeated or prolonged milking. In rat milk, concentrations of transferrin appear to be unaffected. by repeated milking at 4 day intervals,36 but concen~ trations of prolactin decrease to a minimum with 'repeated milking over a 4 hour period. 13 Perhaps repeated milking decreases the secretion of endogenous prolactin into the blood and/or its passage into the milk. In contrast' the amount of DSIP in human milk before nursing was found to be lower than that after a feeding. 28 Stage of Lactation. Considerable variation exists in the relationship between stage of lactation and the concentration of polypeptides in milk, such that it is not possible to describe a general pattern for all. In human milk, insulin is highest in colostrum and declines with progressive lactation; prolactin, calcitonin, growth hormone, TSH and DSIP behave simi-

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larly.2,28,29,37,42-47 Although EGF was found by some workers to decline after parturition,45 other investigators have concluded that milk EGF concentration is not affected by stage of lactation. 21 In contrast, the IGF-I content of human milk increases between early and full lactation (1-6 weeks postpartum).48 Concentrations ·o f relaxin increase in human milk during the first week and remain high up to 8 months of lactation. 49 Similar differences have been observed in animals. In the rat, concentrations of transferrin in milk vary biphasically, decreasing dramatically from days 1-5 but increasing during the latter half of lactation to high levels.36 Extension of lactation by providing dams younger pups to suckle results in an even greater increase in concentrations of transferrin in milk,36 perhaps providing the younger pups with greater amounts of this protein to function in iron absorption or prevention of infection. This increase in concentration of transferrin during extended lactation was identical for experiments with pups of different ages, indicating that the age of the pup is unlikely to be a determining factor. EGF increases in mouse milk steadily from the time of parturition, reaching a peak at day 8 of lactation that is sustained during midlactation and declines with the approach of weaning at 17 days.23,50 Prolactin in rat milk reaches a peak concentration on day 4 that is sustained until day 15, followed by a decline;13,51 the decline may reflect the fall in secretion of endogenous hormone known to occur late in lactation. Concentrations of a salmon calcitonin-like peptide in rat milk are constant throughout lactation,52 but concentrations of immunoreactive GHRH, somatostatin, and growth hormone decrease with time postpartum.51 In cow milk, prolactin concentrations fall with progressive lactation. 53 In porcine milk, concentrations of insulin and neurotensin decrease during transition from colostrum to mature milk,concentrations of prolactin fall more gradually, and the concentration ofbombesin-like immunoreactivity remains relatively constant. 30,54 Observations such as these suggest that varied mechanisms may control the output of different peptides by the mammary gland. A remaining question, as stated earlier, is how changes in milk content of a polypeptide relate to changing needs for the peptide by the suckling infant. Post-conceptual Timing. Differences have been observed in the content of both total and individual proteins in milk from mothers delivering prematurely and at term, but the reasons for these differences are unknown. 1 Among the biologically-active polypeptides, concentrations of insulin are lower in colostrum of mothers delivering prematurely than in those delivering at term. 44 However, no differences were observed in concentrations of EGF in milk of term and preterm mothers.21 Individual Mammary Gland. In humans, discordance of milk protein production has been observed between February 1991 Volume 301 Number 2

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individual mammary glands,55 and in rats higher concentrations of transferrin were found in milk from nonsuckled glands compared with suckled glands.36 However, studies of prolactin concentrations in the milk of the rat did not show significant differences when milk from six simultaneously sampled glands was compared. 13 In lactating sows, wide variation was observed in concentrations of insulin in milk obtained from different teats, but the patterns of change with progressive lactation were similar. 30 Fates of Biologically Active Milk Polypeptides

A potentially important biologically active polypeptide in milk may experience one or more of three fates: it may be degraded, either within milk or the digestive tract of the recipient infant; it may survive digestive processes in an intact form; or it may be processed by digestive mechanisms to yield other biologically active forms. The most traditional fate is degradation to provide dietary substrate in the form of amino nitrogen for growth of the infant. The bulk of the protein in milk undergoes this fate, with gastrointestinal degradation proceeding to yield short peptides that are subsequently absorbed (e.g., as dipeptides).56 Possibly, the original polypeptide might first function biologically either within the mammary gland or within a preceding segment of the gastrointestinal tract in which it survives degradation. A well-established example of this is the milk protein alpha-lactalbumin: before its efficient hydrolysis within the gut lumen, it functions in the mammary gland as a subunit of the enzyme lactose synthetase.3 Some polypeptides may experience degradation in milk even before ingestion. Human colostrum, for example, contains enzymatic activity capable of degrading oxytocin and vasopressin,57 and hydrolysis of prolactin by milk has been observed in vitro.ll Other peptides appear unaffected by incubation with milk. 15.29 At the other extreme, a polypeptide could survive digestive processes and retain its biological activity. Limitations in protein digestive capacity early in postnatal life exist in both man and animals. 58 Moreover, a number of protease inhibitors have been characterized in milk that may protect against peptide degradation in the infant gut.58 Consequently, survival of some dietary polypeptides in an intact form may be favored at this time. Examples include those polypeptides with demonstrable absorption from the gastrointestinal tract upon enteral administration, such as the hormones prolactin, GHRH, TSH, LHRH, TRH, insulin, erythropoietin; neuropeptides such as DSIP; growth factors such as NGF and EGF; or toxins such as that of C. botulinum.2.14.27.58-69 Observations that structural integrity and/or bioligical activity are retained after absorption suggest survival in an intact form but often do not exclude the possibility that a portion of the ingested polypeptide molecule may be degraded. Structural aspects of polypeptides that deTHE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

termine resistance to degradation and capacity for absorption are incompletely defined. 56 The third possible fate of a dietary polypeptide is conversion to an active form by digestive processes, either from an active or an inactive precursor. There are a number of examples of such conversions by digestive proteases, but evidence that they occur in vivo and result in products that may function biologically is limited. Conceivably, processing to an active form could occur in either the infant gastrointestinal tract or within the milk itself. In rat milk, for example, proteolytic activity exists that can cleave the large disulfide loop of prolactin into a form retaining ability to bind to specific receptors. Since this same variant exists naturally in pituitary and plasma, an as yet undefined functional significance is possible. 70 Perhaps the best examples of biologically active polypeptides derived from inactive dietary precursors are those isolated from bovine and human caseins digested with gastrointestinal proteases. 5•71 Examples include a hexapeptide, Val-Glu-Pro-Ile-Pro-Tyr (residues 54-59 of human beta-casein), that was shown to stimulate phagocytosis by mouse peritoneal macrophages and to impart a protective effect against K. pneumoniae infection when injected before challenge with this organism. 71- 73 A tripeptide, Gly-Phe-Leu (residues 60-63 of human beta-casein) showed similar but weaker activity. A dodecapeptide (residues 23-34 of bovine alpha-s-1 casein) and a heptapeptide (residues 177-183 of bovine beta-casein) found in bovine casein hydrolysates have been shown to inhibit angiotensin I-converting enzyme.74.75 They appear to potentiate the action of bradykinin on rat uterus and ileal contraction. A growth-promoting activity for Bifidobacterium bifidus has also been detected in bovine casein hydrolysates. 76 Conceivably, this could limit proliferation of pathogens in the infant intestine by favoring growth of this benign flora. The best characterized biologically active peptides derived from casein hydrolysates are those with opiate activities, the casomorphins and morphiceptin. These include the beta-casomorphins, that vary in length from four to eight amino acid residues and may be derived from both bovine and human beta-caseins. 77- so The beta-casomorphins all share the common N-terminal sequence Tyr-Pro-Phe. When assayed by their ability to inhibit electrically-induced contractions of guinea pig ileum, the human beta-casomorphins are less potent than their bovine counterparts.so Morphiceptin, the amino-terminal tetrapeptide fragment of beta-casomorphin detectable in bovine casein hydrolysates,Sl is 50-100 times more active than beta-casomorphin in receptor binding assays. Beta-casomorphin is also present in commercially available milk and infant formulas. 77 In addition to the beta-casomorphins, opiate peptides have also been derived from bovine alpha-casein by peptic digestion. 82

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That casomorphins may be generated and absorbed in vivo is supported by the detection of beta-casomorphin immunoreactive material in the small intestinal fluid of adult humans83 and minipigs84 after ingestion of cow milk and in the plasma of newborn calve~85 and dogs86 after milk intake. Attempts to demonstrate absorption in adult humans were unsuccessful. 77 A hexapeptide with opiate antagonist activity was recently detected in a peptic digest of bovine kappacasein.87 Attempts to detect another endogenous antiopiate, Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in human28 or rat (Britton JR, Kastin AJ, unpublished observations) milk revealed no immunoreactivity. Biological Roles

A number of possible biological roles have been proposed for milk polypeptides both in the mother and in the recipient infant. However, results supporting these roles are at best suggestive. The presence of high concentrations of some biologically active polypeptides in human milk but not infant formulas 21 ,88 has supported speculation that human milk might confer a biological advantage for the infant by providing a supply of these peptides. In the Mammary Gland. The presence of some biologically active peptides in milk might reflect their role within the mammary gland. This may be especially true for growth factors in milk, some of which may function in growth of the mammary gland.4 Insulin, EGF, and IGFs may all exert mitogenic effects upon mammary tissue, although their precise physiological roles in gland development and function remain to be defined. 4 Human milk also contains · transforming growth factors (TGFs), mitogenic peptides produced by a variety of mammary tumors. 89,90 One factor related to TGF-alpha, designated milk-derived growth factor2 (MDGF-2), may be a candidate for an autocrine or paracrine growth factor produced by and acting within the mammary gland4,89;9o with only secondary appearance in milk. A somewhat larger factor (62 kD) present in milk and mammary tumors but unrelated to TGFs, mammary-derived growth factor-1 (MDGF-1), may also act as an autocrine factor, stimUlating both growth and type IV collagen production by mammary cells.91,92 Milk also contains several growth inhibitory polypeptides. These include TGF -beta90 and a mammaryderived growth inhibitor (MDGI) that inhibits the proliferation of Ehrlich ascites mammary carcinoma cells.93 MDGI-like material is associated with milk fat globule membranes, suggesting that it may be synthesized in mammary epithelial cells and that it exists at least partially in a plasma membrane-associated state.94 Recently, a low molecular weight protein fraction from goat milk has been shown to inhibit casein synthesis by rabbit mammary explants in vitro and to diminish milk accumulation when injected into lactating rabbit teat ducts. 95 The existence of negative autocrine

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control of milk production by this fraction has been proposed. In the Maternal Body. A recently described potential role for milk polypeptides is action at maternal body sites distant from the mammary gland. The best evidence for such a phenomenon is derived from studies of the casomorphins. Material reacting to antisera against both human beta-casomorphin 1-8 and betacasomorphin 1-7 has been detected in the plasma of women during pregnancy and after parturition but not in that of men or non-pregnant women.96,97 More recently, a decreasing concentration gradient ofbeta-casomorphin-8 has been demonstrated between milk and plasma and between plasma and cerebrospinal fluid from lactating women,98 suggesting that biologicallyactive fragments of milk beta-casein may cross the breast parenchyma-blood barrier into plasma and subsequently penetrate the blood-brain barrier to enter the central nervous system. Although a non-mammary source for the immunoreactive material remains to be excluded, it has been suggested that mammary tissue may serve an endocrine function during galactopoesis and that beta-casein could be considered a prohormone.98 The biological role of casomorphins in the central nervous system remains speculative, but an analgesic role is possible. Curiously, these same workers previously observed that a group of women experiencing post-p8rtum psychosis had elevated concentrations of casomorphin in the CSF.99 In Milk. Another possibility is that a milk peptide might function biologically within milk itself. Perhaps the best example of this is kappa casein, which serves to stabilize casein micelles and maintain them and the substantial amounts of calcium and phosphorus they contain in solution. Cleavage of kappa casein in the stomach by the enzyme chymosin leads to the clotting of milk in that organ.58 Milk also contains a variety of protease inhibitors of varying sizes that are believed to inhibit the endogenous proteases also present in mammary secretions.58 In the Infant GastrOintestinal Tract. A variety of roles have been postUlated for milk proteins within the gastrointestinal tract including protection against enteric bacterial infections, provision of digestive enzymatic activities, facilitation of micronutrient absorption, and stimulation of allergic reactions. 1,3 Established gastrointestinal roles for polypeptides in milk are less numerous. Some of the best evidence relates to growth factors in milk, most notably EGF. Enteral administration of EGF to suckling rats results in increased mucosal growth of various regions of the gut, and inclusion of anti-EGF antibody in milk feedings ofsuckiing animals reduces indices of growth.100-102 Such observations have supported the hypothesis that milk EGF may be responsible for at least part of the known enhancement of gastrointestinal growth that accompanies milk feeding of young animals. loo Other growth factors in milk February 1991 Volume 301 Number 2

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may also be involved, however. Gastric administration of bombesin in supraphysiological doses to suckling rats has recently been shown to increase trypsin activity in the lumen of the small intestine103 and to stimulate cell proliferation in gastric and colonic mucosa and exocrine pancreas. 104 Some casein-derived peptides may function within the gastrointestinal tract. A beta-casomorphin analog has been shown to exert an antisecretory effect both in vivo and in vitro on rabbit ileum treated with cholera toxin when administered on the luminal side of the intestine. 105 Absorption of the peptide with subsequent action on opiate receptors on the serosal side appears to be a prerequisite for activity. Since natural betacasomorphins are degraded by intestinal mucosa before transepithelial passage106 and enzymatic activities capable of hydrolyzing casomorphins have been described in the gastrointestinal tract,107 the physiological significance of these observations remains unclear. A casein phosphopeptide has been described that is formed in the distal small intestine of rats fed a casein diet. lOS This peptide is apparently capable of enhancing calcium absorption from the intestine with subsequent increased deposition in bone. Among the peptide hormones, milk TRH could potentially playa role in gastrointestinal motility. This peptide stimulates contraction of the duodenum, a phenomenon that appears on day 3 postnatally but disappears after weaning. 109 In the Body After Absorption. For some polypeptide hormones, a systemic physiological response has been observed after oral administration, supporting the concept of absorption and transfer to their site of action in a biologically-active form. Perhaps the most dramatic example is that of insulin: intragastric administration of insulin to suckling rats resulted in a significant reduction in concentrations of blood glucose.65.66 A hypoglycemic response to intragastric insulin could not be observed after weaning even though a response was found with small intestinal administration of the peptide hormone. These observations suggested that the development of gastric digestive capacity for the peptide was responsible for the lack of an intragastric effect66 and that the low gastric degradation during the suckling period favors survival with consequent absorption and function in the newborn. In the newborn pig, endogenous insulin release is blunted during the first few hours after birth. Nevertheless, the high colostral concentrations of insulin and the permeability of the neonatal pig gut to polypeptides may explain the observed high serum concentrations of insulin at this time. 30 Other examples of systemic biological effects of enterally administered polypeptides were provided by experiments in which feeding of erythropoietin to suckling rats resulted in an increase in reticulocytosis.62.11o Similarly, oral administration ofTSH to suckling rats led to an increase ill T3 and T4 concentrations in THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

serum.64 Intragastrically administered beta-casomorphin has been shown to alter the postprandial release of insulin, somatostatin, and pancreatic polypeptide in adult dogS. 111- U3 Oral administration of supraphysiological doses of NGF to newborn mice elicited hypertrophy of sympathetic superior cervical ganglia,69 and normal newborn rats suckled by a dam producing antiNGF antibody were shown to have smaller sympathetic superior cervical ganglia. 114 For several peptide hormones in milk, even more indirect evidence suggests the possibility of a biological role. Suckling pups whose mothers were subjected to anoxia115 or phlebotomy62.110 exhibited evidence of increased erythropoiesis, suggesting increased erythropoietin in the milk. Injection oflactating rats with TRH was followed by an increase of serum TSH and a decrease in pituitary TSH content in the sucklings,16 suggesting that such injections resulted in increased transfer of TRH by milk with subsequent stimulation of TSH release in the young. Suppression of prolactin in milk by administration of bromocriptine during postpartum days 2-5 resulted in a marked reduction of dopamine turnover in the median eminence and an elevation of concentrations of prolactin in suckled offspring when measured at 30-35 days postnatally.u6 This observation suggested that the normal activity of the temperoinfundibular dopamine system of the rat may be impaired if the concentration of prolactin in milk is reduced during a critical postnatal period. 116 Separation of rat pups from their mothers decreased blood and pituitary gonadotropin concentrations117.l1S and increased available ovarian LHRH binding sites.27 The latter phenomena was reversed by resumption of suckling, and the suckling-induced decline in available LHRH receptors could be prevented by parenteral administration of LHRH antiserum,27 supporting the conclusion that infantile ovarian development in the rat is modulated by the mother by the LHRH in milk. Similarly, the normally high concentrations of growth hormone in the serum of suckling rats fall with fasting and rise again after resumption of milk intake.51 The growth hormone releasing activity of rat milk could only minimally be ascribed to its GHRH content in these experiments.51 Conclusion

It is clear that a large number of biologically active polypeptides exist in the milk of several species. However, information regarding their origin and the factors determining their content in milk is fragmentary. Milk polypeptides may experience several possible fates, including absorption from the infant gastrointestinal tract, although the mechanisms involved are not clear. There are examples of the generation of biologically active peptides by digestive fluids or enzymes, but definitive proof is required that such phenomena occur in vivo and result in products that function physiologically. Biological effects of ingested peptides in the

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suckled young have been shown, although an obligatory requirement for a specific milk-derived peptide in the infant remains to be established. Nevertheless, the presence of such polypeptides in milk and their survival in biologically active forms in both the mother and infant are consistent with the possibility of physiological function in addition to the provision of nutritional substrate. Future exploitation of the techniques of modern molecular biology, such as the use of nucleic acid probes to elucidate sites of milk polypeptide synthesis and of transgenic animals to manipulate milk peptide content,119 may enhance our knowledge of the role of these polypeptides in milk. References 1. Atkinson SA, Lonnerdal B: Protein and non-protein nitrogen in human milk. Boca Raton, CRC Press, 1989, pp. 1-256. 2. Thornburg W, Koldovsky 0: Hormones in milk. A review. J Pediatr Gastroenterol Nutr 6:172-196, 1987. 3. Lonnerdal B: Biochemistry and physiological function of human milk proteins. Am J Clin Nutr 42:1299-1317,1985. 4. Dembinski TC, Shiu RPC: Growth factors in mammary gland development and function. In: Neville MC, Daniel CWo The Mammary Gland. Development, Regulatum, cincl Function. Plenum Press, NY,1987, pp 355-381. 5. Fiat A-M, Jolles P: Caseins of various origins and biologically active casein peptides and oligosaccharides: Structural and physiological aspects. Mol Cell Biochem 87:5-30, 1989. 6. Nickerson SC, Akers RM: Biochemical and ultrastructural aspects of milk synthesis and secretion. Int J Biochem 16:855865,1984. 7. Vonderhaar BK, Ziska SE: Hormonal regulation of milk protein gene expression. Ann Rev Physiol51:641-652, 1989. 8. Bradshaw JP, White DA: Identification of a major N-glycosylated protein of rabbit mammary gland and its appearance during development in vivo. Int J Biochem 12:175'-185, 1985. 9. Lee EY-H, Barcellos-Hoff MH, Chen L-H, Parry G, Bissell MJ: Transferrin is a major mouse milk protein and is synthesized by mammary epithelial cells. In Vitro Cell Dev Biol 23: 221-226, 1987. 10. Gitlin JD, Gitlin JI, Gitlin D: Selective transfer of plasma proteins across the mammary gland in lactating mouse. Am J Physiol230:1594-1602,1976. 11. Grosvenor CE, Whitworth NS: Incorporation of rat prolactin into rat milk in vivo and in vitro. J Endocrinol70:1-9, 1976. 12. McMurtry JP, Malven PV: Experimental alterations of prolactin levels in goat milk and blood plasma. Endocrinology 95: 559-564,1974. 13. McMurtry JP, Malven PV: Radioimmunoassay of endogenous and exogenous prolactin in milk of rats. J Endocrinol61:211217,1974. 14. Werner H, Amarant T, Fridkin M, Koch Y: Growth hormone releasing factor-like immunoreactivity in human milk. Biochem Biophys Res Comm 135:1084-1090, 1986. 15. Strbak V, Alexandrova M, Cacho L, Ponec J: Transport of3HTRH from plasma to rat milk: accumulation and slow degradation in milk and presence of unaltered hormone in gastric content of pups. Biol Neonate 37:313-321,1980. 16. Strbak V, Macho L, AIexandrova M, Ponec J: TRH transport to rat milk. Endocrinol Exper 17:343-350, 1983. 17. Van Noorden S, Heitz P, Kasper M, Pearse AGE: Mouse epidermal growth factor: light and electron microscopical localisation by immunocytochemical staining. Histochemistry 52: 329-340,1977. 18. RaIl LB, Scott J, Bell GI, Crawford RJ, Penschow JD, Niall HD, CoghlanJP: Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature 313:228-231, 1985.

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