Enzymes in Milk: Their Function in the Mammary Gland, in Milk, and in the Infant

Margit Hamosh

Department of Pediatrics, Georgetown University Medical Center, Washington, D.C. 20007

Human milk, like the milk of other species, contains numerous enzymes. Although this topic has been reviewed (1-4), the first two publications provide little information about the physiological significance of these enzymes. Shahani et al. (2) compare the activity level of several enzymes in human and bovine milk, drawing attention to the great differences in the activity levels of numerous enzymes between the two species. The reader is referred to the above-listed reviews, which provide the background for the data reviewed in this chapter. It seems that the best way to approach a discussion of human milk enzymes is to arbitrarily divide the enzymes into three groups: (a) those that function in the mammary gland; (b) enzymes present in milk whose function is un- known; and (c) enzymes that might function in the infant. The latter group would have to remain active during passage through the infant's digestive system. This review does not aim to list or discuss the function of all enzymes in human milk; rather, specific enzymes are selected for discussion of their physiological role as components of human milk (Table 1).

MILK ENZYMES ACTIVE MAINLY IN THE MAMMARY GLAND

Although the physiology of lactation has been studied in experimental animals (in vivo and in vitro, in tissue explants or cell cultures), very little is known about the physiology of lactation in humans. Prepartum mammary secretions and postpartum colostrum and milk can be used as "windows" through which one might obtain information on the function of the human mammary gland in the perinatal period (Table 2). The presence of enzymes in postpartum milk, as opposed to their absence 45 46 ENZYMES IN MILK

TABLE 1. General functions of human milk enzymes

Function Enzyme

Protection (bactericidal) Lysozyme Peroxidase Lipase (LPL, BSSL)a Digestion Amylase Lipase (BSSL) Repair Sulfhydryl oxidase (SOX) Transport (metal carrier) Glutathione peroxidase Alkaline phosphatase Xanthine oxidase Biosynthesis of milk components Phosphoglucomutase Lactose synthetase Fatty acid synthetase Thioesterase Pathogenic Glucuronidase Lipase (LPL, BSSL)

a (LPL) lipoprotein lipase; (BSSL) bile salt-stimulated lipase.

in prepartum secretions, might indicate low levels in the mammary gland before delivery and marked increase in activity after parturition. An example is lipoprotein lipase, the enzyme that controls the uptake of lipoprotein fatty acids from the circulation into the mammary gland. This enzyme plays a key role in the delivery of long-chain fatty acids (7), phospholipids, and cholesterol (14) to the lactating mammary gland. Its absence from prepartum mammary secretions (4) suggests low activity in the mammary gland, an assumption confirmed by low concentrations of fat (1 g/dl) in these secretions (15). A sharp rise in enzyme activity after birth is paralleled by an increase in milk fat concentrations to 3 to 4 g/dl (15,16). A second lipase, the bile

TABLE 2. Milk enzymes that function in the mammary gland

Enzyme Function Ref.

Phosphoglucomutase Galactose synthesis 5 (PGM) Galactosyltransferase Lactose synthesis 6 Lipoprotein lipase Regulates transfer of triglyceride, 7,8 cholesterol, and phospholipid from blood to milk Antiproteases Protection of mammary gland from 9, 10 proteolysis (leucocytes, lysosomes)? 7-Glutamyltransferase Endo and exocytosis of proteins? 11 Xanthine oxidase Secretion of milk fat droplets? 12 Fatty acid synthetase Lipogenesis 13 Thioesterase ENZYMES IN MILK 47

TABLE 3. De novo synthesis of fatty acids in the lactating mammary gland

Product: Fatty acids (mol % fatty acids synthesized) Enzymes

FAS" Thiosterase Species 8:0 10:0 12:0 14:0 16:0 18:0 (ixg/106 cells) (n.g/106 cells) Ref.

Human 4.9 18.3 36.3 22.1 13.1 1.8 2.6 0.03 13 Rat 5.7 29.0 29.0 29.0 15.0 0.8 20.0 0.50 25

a (FAS) fatty acid synthetase. salt-stimulated lipase of human milk, is present in early prepartum secretions (4). This enzyme is known to function in the intestine of the newborn, where it hydrolyzes dietary fat in the presence of bile salts (17,18). It remains to be determined whether this enzyme might also function in the mammary gland before and after parturition, possibly in the intracellular metabolism of fat. The question is, How does the enzyme, which has an obligatory dependence on bile salts (19,20), act in their absence? Although milk contains bile salts (21), the concentration is several orders of magnitude lower than in the intestine. It could, however, be that (a) bile salt concentrations are higher in the mammary gland than in milk, or (b) that specific compart- mentalization of bile salts and lipase might affect their interaction in the cell. On the other hand, higher protein and enzyme concentrations in precolostrum and colostrum, compared to mature milk, might be the result of incomplete tight junctions and of the small volumes secreted before the second or third day postpartum. This is true for amylase (4,22), lysozyme, lactoferrin (23,24), and other proteins, such as IgA (23,24). Mammary secretory cells, present in human milk throughout lactation, can also be used to learn about the function of the human mammary gland during lactation, for example, recent studies on lipogenesis in the lactating human mammary gland. Mammary secretory cells isolated from human milk, were used in these studies (13). The data show that the human mammary gland contains the two enzymes necessary for lipogenesis—fatty acid synthetase and thioesterase II—and that it synthesizes the same type of fatty acids (i.e., C8-Ci6), as do other mammalian species (25). These data are summarized in Table 3. It is possible that the lipogenic system is adaptive and can be repressed by maternal diets of high fat content or stimulated by high-carbohydrate-low-fat diets (26). In general, the enzymes in human milk seem to have a more highly or- ganized tertiary structure than the same enzymes from other sources. This results in greater hydrophobicity of human milk enzymes, possibly account- ing for the remarkable resistance of many enzymes to proteolysis and den- aturation in the infant's gastrointestinal system (3,4). Indeed, there are also 48 ENZYMES IN MILK differences in the rate of disulfide bond formation (i.e., the acquisition of native, biologically active structure by the regeneration of disulfide bonds of denatured, reduced polypeptides) (27), which might explain the function of the potent sulfhydryl oxidase of human milk (28). Recent studies show that this oxidation proceeds slower with a human milk enzyme than with the identical enzyme (lysozyme) of hen egg white, suggesting a high-energy barrier, which would constitute a limiting step (27). It is therefore possible that many milk proteins (enzymes included) might depend on enzyme-catalyzed oxidation of reduced sulfhydryl bonds (29). There is recent evidence that suggests differences in isozymes, degree of glycosylation, and enzyme pattern between identical enzymes in the lactating mammary gland and milk and those in other tissues (3,4).

Phosphoglucomutase

Phosphoglucomutase (PGM) catalyzes the production of glucose-1-phosphate, the first intermediary in the pathway of synthesis of the galactose moiety of lactose. Phosphoglucomutase (a-D-glucose-l,6-diphosphate:a-D- glucose-1-phosphate phosphotransferase) is the product of three loci: PGMi, PGM2, and PGM3. In most tissues, PGMi isozymes account for 85% to 95% of total PGM activity, PGM2 for 2% to 5%, and PGM3 for 1% to 2%. In erythrocytes, PGMi and PGM2 are found in equal amounts, whereas PGM3 is absent. The PGM patterns in human milk are different and independent from those in the erythrocytes and can be explained on the basis of a distinct PGM4 locus (5). The recent study of Cantu and Ibarra (5) is the first report of a distinct gene for a widely distributed protein being functionally restricted to the lactating mammary gland, since no evidence of its activity has been found in other tissues previously studied.

Galactosyltransferase

Galactosyltransferases catalyze the synthesis of the heteropolysaccharide moieties of complex carbohydrates. One of the best known galactosyltransferases is UDP-galactose: iV-acetylglucosamine galactosyltransferase or A protein of the lactose synthetase system. This enzyme has been purified from various animal and human body fluids, but with the exception of the amino acid and carbohydrate content of the bovine milk enzyme, no information about its structure is available. Recent studies show that galactosyltransferases from human amniotic and ascites fluids have similar isoelec- tric focusing patterns, whereas the milk enzyme is less negatively charged (6). The study suggests that the milk enzyme contains less sialic acid, possibly as a result of the neuraminidase activity in human milk (30). This study ENZYMES IN MILK 49 suggests that the electrophoretic difference between the enzyme in milk and in other body fluids is of postribosomal rather than genetic origin. Galactosyltransferases are found in the Golgi membranes of many tissues. The binding of the regulatory protein a-lactalbumin to galactosyltransferase increases the latter's affinity for glucose (from a Km for glucose of 1 M to 1 HIM), thus enabling lactose synthesis at physiological glucose concentrations (31).

Lipoprotein Lipase

Lipoprotein lipase (LPL) regulates the uptake of circulating triglyceride fatty acids, cholesterol, and phospholipids by the lactating mammary gland (7,32,33). LPL has a central role in providing the lipid constituents of milk. In the mammary gland, LPL is located both in the endothelium (its site of activity) and in the alveolar cells, the site of its synthesis. The extreme variation in enzyme levels in the milk of different species (34) suggests either species differences in the mechanism of milk secretion or a possible relationship between damage to the mammary cells and the state of development of the offspring (i.e., the pressure applied to the mammary gland during sucking) (34). Since expression of milk with the aid of a breast pump might cause some rupture of alveolar cells, we have compared the levels of LPL activity in milk expressed from one breast by pumping and in milk that drips spontaneously from the opposite breast (35). Our study shows that LPL levels were lower in pumped than in drip milk. This would suggest that leakage of enzyme from ruptured cells is probably not the mechanism of its release into milk. Recent studies of a patient with familial LPL deficiency (type I hyperlipoproteinemia) show that LPL is absent from milk throughout lactation, suggesting that it is absent also from the mammary gland (36). The authors suggest that a common or closely related genetic locus might be implicated in the normal synthesis of LPL in different tissues. The study further high- lights the key role of LPL in the control of milk fat content and composition. Thus, milk fat concentration was significantly lower in the patient when compared to normal lactating women; furthermore, its composition also dif- fered, the milk containing higher amounts of lauric (C12:0) and myristic (C14:0) acids and considerably less oleic (C18:1) and especially linoleic acid (C18:2). The higher concentration of fatty acids synthesized in the breast tissue (Table 3) is probably due to the restricted fat intake, as well as to much restricted entry of long-chain fatty acids into the mammary epithelial cells (only nonesterified fatty acids would reach these cells from the circulation). Since long-chain fatty acids or their derivatives inhibit fatty acid synthetase (37), their reduced uptake results in enhanced fatty acid synthesis in the mammary gland (36). 50 ENZYMES IN MILK

LPL has no known function in milk or in the newborn but has been implicated in hydrolysis of milk triglycerides and in the release of free fatty acids in human (38) and bovine milk (39) during storage. The increase in milk-free fatty acid concentrations may be associated with the anti-infective activity of human milk (40,41), a phenomenon previously thought to depend only on bile salt-stimulated lipase activity (42).

Fatty Acid Synthetase and Thioesterase

Fatty acid synthetase and thioesterase catalyze de novo synthesis of fatty acids and have recently been described for the first time in human mammary gland secretory cells (Table 3) (13). Indirect evidence suggests that these enzymes may be regulated by the cellular concentration of long-chain fatty acids (26,36).

-y-Glutamyltransferase

•y-Glutamyltransferase activity is high in human colostrum, and although activity decreases thereafter, considerable amounts of enzyme are present in transitional and mature milk (11,43). The enzyme catalyzes the transfer of the -y-glutamyl group, the receptors differing according to the source of •y-glutamyltransferase (the renal enzyme utilizes different amino acid and peptide receptors from the milk enzyme). In addition to kidney, appreciable amounts of 7-glutamyltransferase are present in liver, pancreas, prostate, and breast cyst fluid (43). It has been suggested that the enzyme is localized in the Golgi apparatus and that it plays a role in the endo- and/or exocytotic transport of proteins (11). Indeed, in milk the enzyme is also associated with the membrane fraction of skim milk (44). The high levels of 7-glutamyltransferase in the serum of newborn infants have been attributed to absorption of the intact enzyme from breast milk (43).

Xanthine Oxidase

Xanthine oxidase is a major component of the milk fat globule membrane in bovine milk (1,2). It has been suggested that this enzyme has a function in the secretion of milk fat droplets, possibly by changing the fluidity of the plasma membrane by peroxidizing membrane-associated lipids (12). The very low levels of xanthine oxidase in human milk suggest major differences in the composition of the milk fat globule membrane and possibly in the mechanism of secretion of fat globules into the milk of these two species. In addition to its function in the mammary gland, the enzyme might also act as a metal carrier (Table 4) thereby having also a function in the newborn. ENZYMES IN MILK 51

TABLE 4. Milk enzymes as metal carriers

Glutathione peroxidase: selenium 30% of milk Se Alkaline phosphatase: zinc and magnesium 20% of milk Zn (four atoms per molecule, two essential for activity and two for structure) Mg: two atoms per molecule Xanthine oxidase: iron and molibdenum eight atoms Fe, two atoms Mb per molecule

" Adapted from refs. 45, 46, and 54.

Xanthine oxidase has specific binding sites for iron (eight atoms per molecule), which are important for its enzymatic activity, and for molybdenum (two atoms per molecule) (45,46).

MILK ENZYMES WITHOUT WELL-DEFINED FUNCTION

Although there are many enzymes in human milk whose function is un- known, only two are discussed here.

Lactate Dehydrogenase Similar to many enzymes in milk, lactate dehydrogenase (LD) activity is highest in colostrum and decreases as a function of lactation. Recent studies suggest that in addition to changes in enzyme concentration, there is also a change in isozyme pattern from an LD-5 maximum in colostrum to LD-1 maximum in transitional milk (47). The LD molecule consists of four subunits of two different types, des- ignated H (heart muscle) and M (skeletal muscle). Five different combina- tions of these subunits are possible, corresponding to LD-1 to LD-5. Cardiac muscle is richest in LD-1 and liver in LD-5. The patterns for colostrum and transitional milk differ from that of maternal serum for which LD-2 and LD- 3 are the main isozymes. The change in the isozyme spectrum of the milk enzyme during the early stages of lactation is especially interesting in view of the fact that the LDH isozyme pattern of each organ is considered to be unique.

Plasminogen Activator

Plasminogen activator is another enzyme without a known function. Al- though the enzyme was first reported in milk in 1952 (48), it has only recently been characterized (49,50). The purified enzyme has a molecular weight of 86,000 and was shown to be antigenically different from urokinase, a well- 52 ENZYMES IN MILK characterized plasminogen activator isolated from urine. Inhibition of activity by di-isopropylfluorophosphate (DFP) indicates that serine is at the active site, as in urokinase.

MILK ENZYMES IMPORTANT IN NEONATAL DEVELOPMENT

Proteolytic Enzymes and Antiproteases in Human Milk

Human milk contains both proteolytic enzymes and protease inhibiting activity: The net proteolytic activity will therefore depend on the quantitative interaction between the two proteins (Table 5). Recent studies indicate cas- einolytic activity and elastase-like activity to be present, but no trypsin and chymotrypsin-like activity was detected (9). It is possible that leukocytes are the source of the elastase-like activity in human milk. Although the presence of protease inhibitors in human milk was reported about 30 years ago (3,4), their nature and amounts have been investigated only recently. These studies show that al-antichymotrypsin (3,10) and al-antitrypsin are the main protease inhibitors in human milk. These studies also show that one-third of colostrum specimens studied (18 of a total of 53 samples) had no detectable protease inhibiting activity despite the presence of immunoreactive protease inhibitors; furthermore, caseinol- ytic activity was present in all of these colostrum specimens. The physiological function of protease inhibitors in human milk is not clear

TABLE 5. Milk enzymes that function in the infant

Enzyme Function Ref.

Protease Hydrolysis of milk proteins 2, 9,48 Antiproteases Protect bioactive proteins (enzymes, 2, 10 immunoglobulins) from hydrolysis in milk and in the intestine of the newborn a-Amylase Facilitates digestion of 22 polysaccharides (in milk, formula, and beikost) by the infant BSSL Hydrolysis of fat in the intestine of the 19, 20, 35, 40 newborn; bactericidal activity Sulfhydryl oxidase Catalyzes oxidation of SH groups: 28 possible role in maintaining structure and function of proteins containing disulfide bonds Lysozyme Bactericidal 51,52 Peroxidase Bactericidal; present in leukocytes 53 Glutathione peroxidase Selenium delivery to the infant 54 p-Glucuronidase Breast-milk jaundice? 55 ENZYMES IN MILK 53 at present. We may postulate that they may protect the mammary gland from local proteolytic activity by leukocytic and lysosomal proteases during different stages of differentiation and lactogenesis or during pathogenic conditions such as mastitis; they may prevent the proteolytic breakdown of other enzymes and proteins in milk (and may thus be important in milk banking); and they may affect the absorption of milk proteins (immunoglobulins) in the newborn. Furthermore, the presence of antiproteases would facilitate the delivery of compensatory digestive enzymes (lipase and a-amylase) in an active form from milk to the infant. It has been suggested that the an- titryptic and antichymotryptic activity of human milk may prevent the absorption of endogenous and bacterial proteases in infants and thereby contribute to the passive protection of extraintestinal organs such as the liver (57). The high concentration of antiproteases in colostrum coincides with the period of greatest transfer of nonimmunoglobulin protein from the intestine to the systemic circulation of the newborn (56). Recent studies have determined the primary structure of human p-casein (58) and have suggested that 7-casein is a product of endogenous human milk proteolytic activity (59), as was previously reported for the origin of 7-casein in bovine milk. Small peptides (three to eight amino acids) derived from casein, such as the casomorphines, have specific physiologic activity. Morphiceptin, valmuceptin, and other derivatives are potent and specific agonists of |x-opiate receptors (60). Lindstrom et al. (61) suggest that certain cases of postpartum psychosis may be associated with the presence of these peptides in blood and cerebrospinal fluid (CSF). Whether they affect infant behavior is not known at present.

Sulfhydryl Oxidase

Sulfhydryl oxidase is another enzyme present in human milk (28) and in the milk of other species (3,4) that might function in milk and in the gastrointestinal system of the newborn infant. This enzyme is present in colostrum as well as in mature milk. Sulfhydryl oxidase catalyzes the oxidation of sulfhydryl groups, using O2 as an oxidant and producing equimolar quan- tities of H2O2 and the corresponding disulfide. The enzyme has broad specificity, acting on both small thiol compounds and protein, and might be essential for the activity of proteins whose structure and function depend on intact disulfide bonds (29). The role of this enzyme could involve main- tenance of structural and functional integrity of milk proteins, enzymes, and immunoglobulins. Furthermore, recent reports that this enzyme is stable at low pH (28) suggest that it might retain activity during passage through the stomach and might function in the intestine of the newborn, where it could be instrumental in the uptake of macromolecules by altering the physical state of the intestinal mucus diffusion barrier (28). Recent studies show that 54 ENZYMES IN MILK

TABLE 6. Some characteristics of milk enzymes active in the infant

Enzyme Sulfhydryl Characteristic Amylase BSSL oxidase

Distribution in milk Aqueous phase Aqueous phase Skim milk membrane Effect of milk storage Stable Stable Stable (-20°C, -70°C) Stability to low pH pH > 3.0 pH > 3.0 pH > 3.0 (passage through stomach) Presence in preterm (PT) Equal activity PT Equal activity PT ? and term (T) milk andT andT Pattern through Colostrum greater Colostrum equal ? lactation: than milk to milk precolostrum, colostrum, milk pH optimum 6.5-7.5 7.4-8.5 7.0-7.5 Evidence of activity in Yes Yes Yes intestine Presence in milk of other ?? Primates and Rat, cow, species carnivores rabbit

sulfhydryl oxidase is present in skim milk membranes and that it is resistant to storage at -20°C (44) (Table 6). a-Amylase

The newborn infant has adequate levels of lactase, an intestinal brush border enzyme that hydrolyzes lactose, the main carbohydrate of human milk; however, a-amylase, the chief polysaccharide-digesting enzyme is not developed at birth, even in full-term infants. a-Amylase in the duodenum amounts to only 0.2% to 0.5% of the adult level (62). Human milk contains 1.2 to 1.5 g/dl oligosaccharides ranging in chain length from penta- to tetra- decasaccharides (63). In addition, many breast-fed infants receive glucose polymers or starch from infant formulas or beikost, given often as supple- ments. For the digestion of glucose polymers, the infant depends on salivary amylase (62), glucoamylase (64), and the amylase of human milk (22). Recent studies show that amylase is present in milk secreted by mothers of preterm and term infants (22). Enzyme levels remain high throughout lactation, and activity is stable during storage for months at either -20°C or -70°C (4). a-Amylase is resistant to low pH values (down to 3.0-3.5) (22) and maintains activity in the newborn's intestine (65) (Table 6). During the past 50 years, there has been an increasing tendency to intro- ENZYMES IN MILK 55 duce solid foods into the infant's diet as early as the first month of life. Such early addition of starch might be well tolerated in breast-fed infants because of high intestinal levels of a-amylase provided by human milk. Indeed, this assumption is supported by observations in Egypt of a lack of digestive disturbances in breast-fed infants offered starch-containing foods early, a practice that occurs particularly among the poor (2).

Bile Salt-Stimulated Lipase

Because of very low pancreatic lipase, the newborn infant depends mainly on lingual, gastric, and human milk bile salt-stimulated lipase (BSSL) for the digestion of dietary fat (66-69). Hydrolysis of milk fat involves first the penetration of lingual (66) and gastric (67) Upases into the milk fat globule and partial hydrolysis of the core triglyceride (68). This process is then completed in the intestine by the BSSL of human milk. The combined activity of these two enzymes can lead to complete hydrolysis of dietary fat and can thus effectively substitute for low levels of pancreatic lipase. The properties of human milk BSSL are fully compatible with activity in the intestine of the newborn (Table 6). The enzyme is stable at low pH and thus not inactivated in the stomach and remains active in the intestine for at least 2 hr (20). Its lack of substrate specificity and its ability to hydrolyze triglycerides completely to free fatty acids and glycerol suggest efficient hydrolysis of triglycerides and various esters (such as retinyl palmitate, the main component of vitamin A) (18). BSSL is present in precolostrum and maintains high activity in milk throughout lactation (4,35). Activity is stable during storage at either — 20°C or -70°C, indicating that banked milk maintains its fat-digesting potential (70). As mentioned earlier, the enzyme might contribute to the antiprotozoan (Giardia lamblia) activity of human milk (40,42). Interaction between lactoferrin (present in high concentrations in human milk) and BSSL has recently been reported (71). This interaction leads to a 1.4-fold increase in hydrolytic activity, suggesting that lactoferrin might be the milk factor previously thought to stimulate milk BSSL activity (72). Contrary to earlier reports (18), we have recently found high BSSL activity in the milk of carnivores (both terrestrial and aquatic) (73,74), indicating that the enzyme is not limited entirely to primate milk. This will enable studies on the biology of the enzyme not only in milk but also in the mammary gland.

Glucuronidase

Breast-fed infants have a higher incidence of jaundice than formula-fed infants (75). Breast-milk jaundice, due to prolonged nonconjugated hyper- 56 ENZYMES IN MILK bilirubinemia, was attributed initially to inhibition of hepatic UDP-glucu- ronyltransferase by the steroid pregnane-3-7-20-p-diol (76) and, subse- quently, to high levels of free fatty acids in jaundice-inducing milks (77). However, no association was found between milk lipase levels and free fatty acid concentrations (78). A recent study shows an association between high P-glucuronidase activity in milk and breast-milk jaundice (55). Glucuroni- dase cleaves glucuronic acid from bilirubin glucuronide, liberating unconjugated bilirubin, which is more easily absorbed from the intestine than the conjugate.

Lysozyme

Lysozyme catalyzes the hydrolysis of the (1-4) linkage between N-acetylglucosamine and Af-acetylmuramic acid in bacterial cell walls. The enzyme lyses mostly gram-positive and a few gram-negative bacteria; it is a major component of the human milk whey fraction and has been shown to play a role in the antibacterial activity of human milk. The lysozyme of human milk is composed of 130 amino acid residues and has a molecular weight of 14,400 daltons. Although its sequence exhibits considerable ho- mology with the lysozyme of chicken egg white (51), recent studies show a marked difference in the tertiary structure of the two proteins, resulting in greater organization and hydrophobicity of the human milk protein (52). Lysozyme and ot-lactalbumin of human milk seem to be derived from a common ancestor molecule on the basis of identical amino acids in 49 po- sitions (79). Although not related to the topic of enzymes in human milk, it is worth noting that another antimicrobial agent in human milk, lactoferrin, was recently shown to be highly resistant to proteolysis both in its iron-free (apolactoferrin) and iron-containing forms (80,81). Because native milk lactoferrin is largely free of iron, it can withold iron from, and thus, retard, the in vitro growth of microorganisms (82). The marked susceptibility of bovine milk lactoferrin to proteolysis led investigators to suggest that the unusual resistance of human apolactoferrin to proteolysis may reflect an evolutionary development designed to permit its survival in the intestine of the infant (80).

Peroxidase •<

Peroxidase in human milk was earlier considered to be lactoperoxidase (83), but a recent study established that the activity in milk is derived from milk leukocytes and is thus a myeloperoxidase (53). The distinction between a true secretory peroxidase (lactoperoxidase) and a peroxidase derived from leukocytes (myeloperoxidase) is important, because the two enzymes have different structures (single polypeptide chains of molecular weight 77,000- ENZYMES IN MILK 57

79,000 versus two subunits with a molecular weight of 118,000 and 144,000, respectively). Both enzymes catalyze the oxidation of thiocyanate ions to products with bacteriostatic activity; however, only myeloperoxidase catalyzes the oxidation of the chloride ion; the products of the latter reaction only have bactericidal activity. It is interesting that bovine milk and human saliva have potent lactoperoxidase activity. The difference in the nature and source of human milk peroxidase reported by the two investigators can be attributed to the different methods used, the second group (53) employing more sensitive assay techniques.

Alkaline Phosphatase

Alkaline phosphatase is located on the luminal surface of the epithelial cells of the ducts and acinar glands. The enzyme is released into milk as part of the plasma membrane during the formation of milk fat globules. The high levels of the enzyme in colostrum and intermediate milk may be due to the sudden activation of the milk secretory mechanism. Although the enzyme was purified from bovine milk about ten years ago (4), it has only recently been characterized in human milk (84,85). The conclusions reached by two groups of investigators differ as to the nature of the enzyme in milk. Whereas one group (84) suggests that functional, antigenic, and structural analysis indicate that the milk enzyme is the same protein species as that of adult human liver, the data reported by the second group (85) suggest that the milk enzyme is a mixture of isozymes similar to bone and liver alkaline phosphatase. Alkaline phosphatase is a metal-carrying enzyme (Table 4) (46), and it contains four atoms of zinc per molecule, two essential for its enzymatic activity and two fulfilling a structural role. In addition to zinc it also contains two magnesium atoms. Much remains to be learned about the function of many milk enzymes. It is important to know their origin, mechanism of secretion into milk, com- partmentalization among the various milk fractions as well as whether their activity changes as a function of length of lactation and pregnancy.

ACKNOWLEDGMENTS

Supported by NIH Grant HD-20833. The expert secretarial help of Ms. Barbara Runner is gratefully acknowledged.

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