ARTICLE IN PRESS International Dairy Journal 16 (2006) 533–545 www.elsevier.com/locate/idairyj Review Physiological role of indigenous milk enzymes: An overview of an evolving picture Nissim Silanikovea,, Uzi Merinb, Gabriel Leitnerc a b Department of Ruminant Physiology, Institute of Animal Science, A.R.O., The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel Department of Food Science, Institute of Technology and Storage of Agricultural, Products, A.R.O., The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel c National Mastitis Reference Center, Kimron Veterinary Institute, P.O.B. 12, Bet Dagan 50250, Israel Received 29 May 2005; accepted 26 August 2005 Abstract Over 60 indigenous enzymes have been identiﬁed so far in the milk of various mammalian species. The vast majority of research in this area has focused on their use as indicators of processing (mainly pasteurization), contribution to dairy product quality and investigating the factors that affect their level in milk. The aim of this article is to provide an overview of data accumulated during the last 5 years, mostly for bovine and human milk, which shows that milk indigenous enzymes play a key role in regulating lactogenesis, e.g., inducing active involution, and that they are essential components of antioxidation and the innate immune system of milk. r 2006 Elsevier Ltd. All rights reserved. Keywords: Milk; Colostrum; Indigenous enzymes; Review; Physiological role Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Physical nature of milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Distribution of milk enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Factors governing the level of enzymes in milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Physiological roles of milk enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Role of milk enzymes as pro-digestive factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Role of the plasmin system in the regulation of mammary secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Role of the plasmin system in induction of involution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. The plasminogen activator–plasminogen–plasmin-based negative feedback mechanism as a working hypothesis 2.5. Milk enzymes as part of the innate immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533 533 534 535 536 537 537 537 539 540 541 542 542 1. Introduction 1.1. Overview Corresponding author. Tel.: +972 8 9484436; fax: +972 8 9475075. E-mail address: [email protected] (N. Silanikove). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.08.015 Milk synthesis starts in the epithelial cells of the mammary gland at the end of pregnancy to support the ARTICLE IN PRESS 534 N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545 nutrition and promote the health of the off-spring. Milk contain vital nutrients such as proteins, carbohydrates, lipids, minerals and vitamins, together with bioactive substances including immunoglobulins, peptides, antimicrobial factors, hormones and growth factors (Clare & Swaisgood, 2000; Grosvenor, Picciano, & Baumrucker, 1993). Over 60 indigenous enzymes have been identiﬁed so far in the milk of various mammalian species (Fox, 2003). Because of its complex composition, lactogenesis requires the concerted action of many transport processes and the presence of many milk constituents may have biological effects on both the mother (Silanikove, Shamay, Shinder, & Moran, 2000), and the survival of her off-spring (Koldovsky, 1998). The purpose of the present review is to provide an overview of data, mostly on bovine and human milk, showing that indigenous milk enzymes play a key role in regulating lactogenesis, including inducing active involution, and that they are essential components of antioxidation and the innate immune system of milk. A brief review of literature regarding the physical nature of milk, the distribution of enzymes between milk compartments and factors governing their level in milk is presented initially as a background. 1.2. Physical nature of milk Though its structure appears to be continual and homogeneous, milk is composed of at least ﬁve physically and functionally discrete phases (Fig. 1): Whey: Milk serum, commonly known as whey, is the medium in which all compartments are homogeneously dispersed. Whey is composed of water in which minerals, various organic molecules, proteins and peptides are dissolved. Fat globules: The fat is dispersed in milk as small droplets that are enveloped by a plasma membrane rich in phospholipids and commonly known as the milk fat globule membrane (MFGM). In bovine milk, these Fig. 1. Schematic representation of physical phases of milk. The area between the milk particles represents the milk serum (whey), the phase in which all other phases are homogenously dispersed. globules range in size from 1 to 8 mm and average 3–4 mm in diameter (Heid & Keenan, 2005). Casein micelles: The main protein in milk is casein, of which there are 4 types in bovine milk, arranged as large colloidal particles commonly known as micelles. Highresolution ﬁeld-emission scanning electron microscope micrographs of casein micelles revealed spherical particles in the range of 200 nm; the surface of the micelle is not smooth but contains gaps between the tubular substructures (Dalgleish, Spagnuolo, & Goff, 2004). Membrane vesicles: The secretion of various membrane vesicles into the extracellular space is a frequent phenomenon described in normal and tumor cells (Hugel, Carmen Martinez, Kunzelmann, & Freyssinet, 2005). Two types of vesicles are secreted by cells. Exosomes, typically 40–100 nm in diameter, originate from endocytic multivesicular bodies, and are released in an exocytic manner. Although the functions of exosomes remain largely unresolved, they are thought to play immunoregulatory and antitumoral roles (Fevrier & Raposo, 2004). Microvesicles, with a diameter in the range 100–1000 nm, originate from the cell surface membrane and are shed directly into the extracellular space, a process that seems to be important for membrane turnover, tumor ganglioside metabolism and vascular regulation (Hugel et al., 2005). The processes of exosome secretion and membrane shedding are poorly understood. In bovine milk, 40–60% of the membranous phospholipids are in the skim milk, the remainder being associated with the MFGM (Huang & Kuksis, 1967; Morton, 1954; Plantz & Patton, 1973). The skim milk membrane vesicles comprise approximately 1% of the total milk lipids (Huang & Kuksis, 1967) and 5%, w/w, of the total milk proteins (Morton, 1954). Morton (1954) was the ﬁrst to show that normal cows’ milk contains enzymatically active lipoprotein particles, which he called ‘milk microsomes’. Based on their size (30–200 nm), enzymatic nature and chemical composition, it was concluded that milk microsomes originate directly from the microsomes of secretory epithelial cells (Bailie & Morton, 1958a, b). Thus, milk microsomes most likely reﬂect the secretion of endosomes (i.e., vesicles that originate deep in the cell) from mammary epithelial cells. The possibility that microvesicles are also released into the milk by shedding fragments of the apical membrane of the mammary epithelial cells should be taken into consideration and is supported by some histological data, which show budding of microvilli (Wellings, Deome, & Pitelka, 1960), and the presence of detached microvilli in the ‘ﬂuff’ (membranous) fraction of skim milk (Plantz & Patton, 1973). The secretion of microvesicles into milk may also function in regulating homeostasis of the apical membrane of the mammary epithelial cells (Kanno, 1990). Intracellular vesicles are constantly fused into the apical membrane during exocytotic secretion of milk proteins, lactose, calcium and other components of the aqueous phase of milk (McManaman & Neville, 2003), ARTICLE IN PRESS N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545 while cytoplasmic lipid droplets that move to the apical membrane are enveloped by apical membrane and secreted as membrane-bound milk fat globules (Wu, Howell, Neville, Yates, & MacManaman, 2000). In general, the composition of phospholipids derived from milk vesicles and the MFGM are very similar (Huang & Kuksis, 1967), which points to a common origin of the two types of membrane (Patton, Mccarthy, & Durdan, 1966). Shennan (1992) found that K+ and Cl ions crossmembrane vesicles by conductance pathways. The similarity between ion transport by skim milk membrane vesicles and by the apical side of the intact mammary epithelium suggests that the former may be a good model in which to study solute transport by the apical membrane of mammary secretory cells. Silanikove et al. (2000) have identiﬁed a casein-derived peptide that blocks K+ uptake into skim milk membrane vesicles and demonstrated that this peptide down-regulates milk secretion in vivo. Thus, skim milk membrane vesicles appear to maintain their integrity and functionality during storage in the mammary gland. Milk cells: The milk of various mammals contains a heterogeneous population of cells, commonly referred to as somatic cells (SC). In most species, the predominant cells are leukocytes, composed of lymphocytes, polymorphonuclear neutrophils (PMNs) and macrophages, which serve as important components in the mammary defense against potential pathogens, mostly bacteria (Paape, Bannerman, Zhao, & Lee 2003). In bacteria-free cows’ milk, macrophages (Concha, Holmberg, & Astrom, 1986) and epithelial cells (Leitner, Shoshani, Krifucks, Chaffer, & Saran, 2000) are the predominant cell type (35–79%). Following detection of an invasion of pathogens into the mammary gland, macrophages release chemo-attractants, which trigger the migration of PMNs from the blood toward the infection in the gland, increasing their proportion from a basal level of 5–25% to approximately 90% of the total cell population. PMNs phagocytose the invaders and then destroy them by using reactive oxygen species and a range of proteolytic enzymes (Paape, Wergin, Guidry, & Pearson, 1979). 1.3. Distribution of milk enzymes Milk is not a homogeneous solution of enzymes; rather, given enzymes are speciﬁcally associated with one or more of the above-described ﬁve distinct phases (see Table 1 in Shahani, Harper, Jensen, Parry, & Zittle, 1973). The distribution of enzymes in milk most likely reﬂects the way in which they were secreted into the milk and their tendency to associate with particular milk constituents or phases. Little is known about the mechanisms by which indigenous enzymes enter milk, but knowledge of these processes is important for understanding their physiological role. The term ‘spilling over’ from epithelial mammary cells or serum during milk secretion was used in earlier 535 (Kitchen, Taylor, & White, 1970) and later (Farkye, 2003) studies as an explanation for their presence in milk. However, it is doubtful if the term ‘spill over’ is justiﬁed in explaining the origin of enzymes in milk. Milk components are secreted through organized pathways (McManaman & Neville, 2003) without evidence for spillage or waste of cytoplasm during the process, except for caprine milk, which contain a lot of cytoplasmic particles (Neveu, Riaublanc, Miranda, Chich, & Martin, 2002). In some cases, there is an association of crescent material with 1% of the MFGM in bovine milk (Huston & Patton, 1990). A variable but low level of crescent material was also found in the milk of other mammals, such as goats, rats, pigs, sheep, rabbits and humans (Janssen & Walstra, 1982). According to Wooding (1977), these crescents contain trapped cytoplasma, and therefore may contain cellular enzymes. However, Huston and Patton (1990) suggested that an abnormality of the inner protein coat of the MFGM may be responsible for crescent formation and, in that case, the crescent would be expected to be a poor source of enzymes. In any event, there is no reason to assume that the secretion of crescents is an unregulated phenomenon. In the mammary gland, the tight junctions of the alveolar epithelial cells are impermeable even to small atoms or molecules such as Na+ and K+ (Nguyen & Neville, 1998). It should also be borne in mind that tight junctions separate milk and extracellular ﬂuid, in which the concentration of proteins is much lower than in the blood serum. Nevertheless, it is commonly believed that the source of albumin in milk is blood serum and abbreviations such as human serum albumin (HSA) and bovine serum albumin (BSA) are commonly used to describe the albumin in milk. Shamay et al. (2005) have shown recently that the mammary gland itself is a source of milk albumin. The increase in the level of albumin in milk was previously taken as evidence for disruption of the tight junctions (Nguyen & Neville, 1998). However, Shamay et al. (2005) showed that, as part of the innate immune non-speciﬁc defense system the synthesis and secretion of albumin increased under conditions that typically cause disruption of tight junctions. Thus, if blood serum is a source of a given milk enzyme, it is probably through dedicated transcellular transport systems, such as that responsible for the transfer of immunoglobulins (McManaman & Neville, 2003) The largest group of indigenous milk enzymes is that associated with the MFGM and vesicle membranes (Shahani et al., 1973). This phenomenon probably reﬂects the cellular sources of these membranes, i.e., Golgi membranes (Powell, Jarlfors, & Brew, 1977), the rough endoplasmic reticulum (RER) (Jarasch, Bruder, Keenan, & Franke, 1977) and plasma membranes (Dowben, Brunner, & Philpott, 1967). A prominent marker of Golgi membranes from bovine mammary gland epithelial cells is galactosyltransferase (Keenan, Morre, & Huang, 1972), which also has high speciﬁc activity in the MFGM (Powell et al., 1977). A b-type cytochrome which is speciﬁcally ARTICLE IN PRESS 536 N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545 associated with the RER in cows has also been identiﬁed in MFGM and milk microsomes (Jarasch et al., 1977). The ‘sidedness’ in the membrane is also important in considering the physiological role and function of membraneassociated enzymes. Enzymes located within the inner side of MFGM or milk vesicles are intact and therefore their activity would be latent in respect to the outer membrane environment. Patton and Trams (1971) showed that nucleotide pyrophosphatase is conﬁned largely to the inner membrane surface, 50 -nucleotidase is located on the outer surface and Mg2+-activated ATPases are in both surfaces. In uninfected glands, sporadic invasion of bacteria into the gland is counteracted by macrophages, which phagocytose and digest them intracellulary (Paape et al., 2003). Thus, in the uninfected udder, leukocyte enzymes most likely do not interact with milk proteins. Intramammary infection causes an elevation of the somatic cell count (SCC), mostly through increased number of PMNs, associated with increased proteolytic activity in milk (Leitner, Chaffer et al., 2004; Leitner, Merin, & Silanikove, 2004). There is evidence that the oxidative stress associated with clinical mastitis induces massive liberation of proteolytic enzymes from PMNs, which in turn affect proteolysis and cause damage to the mammary tissue (Le Roux, Laurent, & Moussaoui, 2003). Milk from mastitic cows is not used by the dairy industry. However, udders of 30–50% of cows in herds in modern dairy systems may be subclinically infected and this milk is collected routinely by the industry. An important question which arises is whether leukocyte enzymes, under subclinical conditions, affect milk components. PMNs contain a number of lysosymal proteolytic enzymes, including neutral and acidic proteases, elastase and cathepsin B and D (Considine, Healy, Kelly, & McSweeney, 2004). During inﬂammation, there is also increased secretion of lysosomal enzymes, such as N-acetyl-b-D-glucosaminidase (NAGase) from epithelial cells (Leitner, Chaffer et al., 2004; Leitner, Merin, Silanikove, Ezra et al., 2004). Therefore, it would be difﬁcult to distinguish between leukocyte-derived and epithelial cell-derived enzymes during subclinical mastitis. 1.4. Factors governing the level of enzymes in milk The composition of milk is adapted to support the demands of the off-spring during the pre-weaning period, which is reﬂected in great interspecies variability in the gross composition of milk. Limited information, particularly comparison between human and cows, suggests that signiﬁcant inter-species differences exist also in the level of enzymes in milk (Table 1 in Shahani, Kwan, & Friend, 1980). In general, the level of enzymes in human milk is much greater than in bovine milk, e.g., adenosine triphosphatase ( 23), alanine aminotransferase ( 400), a-amylase ( 40) lysozyme ( 100) and NAGase ( 3000). However, in certain cases, the level of particular enzymes are much higher in bovine than in human milk, e.g., lactoperoxidase ( 100), alkaline phosphatase ( 40) and xanthine oxidase ( 10). Peroxidase activity in llama milk is less than 10% that in bovine or ovine milk but NAGase activity is over 20-fold higher; the activity in llama milk of both enzymes resemble more closely their activities in human milk than bovine milk (Morin, Rowan, & Hurley, 1995). In general, the enzyme content of human or ruminant colostrum is higher from that in corresponding mature milk (Shahani et al., 1973) or in ruminant milk (Shahani et al., 1980). Some milk enzymes, such as lactic dehydrogenase and malic dehydrogenase, may reﬂect the activity of epithelial cells. Kjellber and Karlsson (1967) found that the zymogram pattern of lactic and malic dehydrogenase in milk is different from that in blood serum, strongly suggesting that they are synthesized in the mammary gland. These authors found also that the activity of these two enzymes is inversely related to body weight, e.g., in mouse, human and cow, in agreement with the higher metabolic rate in cells from small animals than large ones. Grigor and Hartmann (1985), who compared the activity of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, malic enzyme, lactate dehydrogenase and malate dehydrogenase in the milk of sows, rats and rabbits, concluded that the activity of these enzymes accurately reﬂects enzyme activity in the epithelial cells of the mammary gland. The activity of lactic dehydrogenase in milk increased by a factor of 2–3 between parturition and peak milk production, whereas other enzymes, in particular glucose 6-phosphate dehydrogenase, may increase by up to 20-fold between these times (Gul & Dils, 1969; Richards & Hilf, 1972, in rat; Gumaa, Greenbaum, & McLean, 1973, in sheep). Thus, it may be concluded that some milk enzymes are constitutive components of milk, while others are induced at particular periods of the lactation cycle. In cows, the marked increase in secretion of NAGase in response to inﬂammation, provides a sensitive marker for the detection of mastitis (Pyorala, 2003). Shahani et al. (1980) reviewed some data showing that increased dietary fat intake in lactating women increased the levels of lipase, esterase and alkaline phosphatase, enzymes that play a role in digestion, assimilation and metabolism of fat; whereas protein supplementation of malnourished women increased alkaline phosphatase and xanthine oxidase activity in their milk. Neville, Waxman, Jensen, and Eckel (1991) presented data suggesting that lipoprotein lipase in human milk is regulated by plasma insulin, similarly to adipose tissue. Expression of plasma membrane Ca2+-ATPase type 4b in MFGM was lower in cows that developed milk fever than in control cows (Prapong, Reinhardt, Goff, & Horst, 2005). It may be concluded that the levels of enzymes in milk are species-speciﬁc, and are affected by the metabolic activity of cells, stage of lactation, whether the enzyme is secreted in constitutive or inductive manner and the hormonal, nutritional and metabolic status of the producing animal. ARTICLE IN PRESS N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545 2. Physiological roles of milk enzymes 2.1. Role of milk enzymes as pro-digestive factors Human milk provides digestive enzymes (amylase and lipase) that compensate the newborn for immature pancreatic functions (Hamosh, 1998). In breast-fed infants, digestion of milk triglycerides, the major source of energy and long-chain polyunsaturated fatty acids, is catalyzed by the concerted action of gastric lipase, colipase-dependent pancreatic lipase and bile salt-stimulated lipase (BSSL). The major proportion of BSSL is present in the milk and a lesser part originates from the infant’s exocrine pancreas (Hernell & Blackberg, 1994). Human milk BSSL hydrolyses ceramides and may thus have a role in sphingomyelin digestion, but only after initial hydrolysis to ceramide and phosphorylcholine. Thus, both sphingomyelinase and BSSL may be important for optimal use of human milk sphingolipids (Nyberg et al., 1998). BSSL is also present in milk from dog and cat (Freed, York, Hamosh, Sturman, & Hamosh, 1986) and ferret (Sbarra et al., 1996), suggesting that it may be important also in non-primate species. It is well known that colostrum is an important source of immunoglobulins for the neonate. Studies with cows show that colostrum intake inﬂuences plasma enzyme activities, either because colostral enzymes are absorbed, or as a consequence of endogenous production, which for some enzymes appears to be modiﬁed by the time of ﬁrst colostrum intake (Zanker, Hammon, & Blum, 2001). While the absorption of colostral g-glutamyltransferase has been conﬁrmed amply (Zanker et al., 2001), it is not fully clear whether this is also the case for alkaline phosphatase and aspartate-aminotransferase. The physiological importance, if any, of the absorbed enzymes in the neonatal calf is not clear at present. 2.2. Role of the plasmin system in the regulation of mammary secretion Milk secretion and mammary function are regulated by local mechanisms sensitive to the frequency and efﬁciency of milking (Daly, Owens, & Hartmann, 1993; Wilde & Peaker, 1990). Acute local control of milk secretion occurs through autocrine feedback inhibition by milk-borne factors. Sustained changes in the frequency of milking and milk secretion are associated with longer-term adaptation in the degree of differentiation and, ultimately, the number of mammary epithelial cells. Differentiation of cultured mammary cells is suppressed by a milk fraction containing the inhibitor, suggesting that intra-mammary regulation of differentiation in vivo is elicited by the same autocrine regulator subsequent to its acute effect on milk secretion. This autocrine factor may affect mammary cell differentiation by modulating the number of cell surface hormone receptors for prolactin, thereby changing their sensitivity to circulating hormones (Wilde & Peaker, 1990). In addition to the regulation by milking, milk secretion 537 also depends on external factors, such as emotional stress, as well as harsh physical conditions such as heat stress and water deprivation (Silanikove, 2000). The fast modulation of milk secretion in response to these challenges may point to a similar mechanism of both phenomena (Silanikove et al., 2000). Wilde, Addey, Boddy, and Peaker (1995) presented evidence suggesting that local regulation of milk secretion by milk removal is through autocrine feedback inhibition by a single goats’ whey protein of M(r) 7600, which they termed FIL (Feedback Inhibitor of Lactation). However, despite the fact that a decade has passed since this work was published, there is no information regarding the complete amino acid sequence of FIL or identiﬁcation of the gene coding it. This information is critical for veriﬁcation of the concept. The involvement of the plasminogen activator (PA)– plasminogen–plasmin system in many biological phenomena reﬂects the ubiquitous presence of plasminogen in biological ﬂuids and the ability of numerous cell types to synthesize, in a highly regulated manner, PA, and inhibitors of plasmin and PA. This system is particularly intensively studied in respect to its role in the lysis of blood clots (Sidelmann, Gram, Jespersen, & Kluft, 2000) and regulation of cell activity and cell attachment in various tissues (Saksela & Riﬁrin, 1988). Plasmin, the principal proteolytic enzyme in milk, is found mainly in its inactive or zymogen form, plasminogen. The conversion of plasminogen to plasmin is modulated by PA (Politis, 1996), two types of which exist in mammals: urokinasetype PA (u-PA) and tissue-type PA (t-PA) (Heegaard, Rasmussen, & Andreasen, 1994; Politis, 1996). Plasmin, plasminogen, and t-PA are closely associated with the casein micelles (Politis, 1996), whereas u-PA is associated with neutrophils (Politis, Voudouri, Bizelis, & Zervas, 2003; Politis, Zavizion, Cheli, & Baldi, 2002) and inhibitors of PA and plasmin are in the milk serum (Precetti, Oria, & Nielsen, 1997; Politis, 1996). The close proximity of plasmin to its substrate ensures that hydrolysis is an efﬁcient process (Korycka-Dahl, Dumas, Chene, & Martal, 1983) and it is the primary agent of proteolysis in goodquality milk (Kelly & McSweeney, 2003). The association of t-PA with the casein micelle suggests that it plays a pivotal role in regulating plasmin activity under nonpathological conditions. However, during bacterial infection and the increase in neutrophil numbers in the mammary gland, u-PA activity increases (Leitner, Chaffer et al., 2004; Zachos, Politis, Gorewit, & Barbano, 1992). Plasmin preferentially cleaves polypeptide chains after a lysine or, to a lesser extent, an arginine residue (Ueshima, Okada, & Matsuo, 1996). b-CN is the preferred substrate for plasmin and its hydrolysis results in the production of g-caseins and proteose–peptones (Andrews, 1983). aS1-Casein (McSweeney, Olson, Fox, Healy, & Højrup, 1993) and aS2-casein (Le Bars & Gripon, 1989) are also susceptible to proteolysis by plasmin and the l-caseins are products of hydrolysis of aS1-casein (Aimutis & Eigel, ARTICLE IN PRESS N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545 538 1982). However, k-casein is resistant to proteolysis by plasmin (Diaz, Gouldsworthy, & Leaver, 1996). The insulin-like growth factor binding proteins (IGFBPs) have been shown to interact with several proteins present in milk including as2-casein, lactoferrin and transferrin. These interactions implicate the IGFBPs in the regulation of plasminogen activation since plasminogen and t-PA also bind to as2-casein (Flint et al., 2001) (Fig. 2). There is evidence for the existence of a close association between the PA–plasminogen–plasmin system and gradual involution (the decline phase of lactation). Increased plasmin and PA activity in bovine milk are correlated with gradual involution (Politis, 1996). Treatment with bovine somatotrophin prevents the increase in plasmin during gradual involution, indicating that bovine somatotrophin interferes with the conversion of plasminogen to plasmin (Politis, 1996). Brown, Law, and Knight (1995) found that relative amounts of g-caseins were highly negatively correlated with milk yield in the declining phase of lactation, reﬂecting the gradual involution of the gland at this time. The counter-part of g-casein is the N-terminal fraction b-CN f (1–28) (Andrews, 1983), and therefore this peptide should also be highly negatively correlated with milk yield in the declining phase of lactation. Thus, the data of Brown et al. (1995) support the concept that b-CN f (1–28) plays a role in the regulation of milk secretion (Silanikove et al., 2000). Milk volume is determined by osmotic-coupled water ﬂow. In bovines, the secretion of K+, Na+ and Cl determines approximately 40% of the driving force, with the rest being determined by lactose (Shennan & Peaker, 2000). The currently held view is that lactose and monovalent ions are secreted into the lumen of the mammary gland mainly via vesicles (Shennan & Peaker, 2000). However, a direct contact between monovalent ions inside the epithelial cells and ﬂuid stored in the lumen of the gland is possible, since the apical membrane of the epithelial cells contains K+, Na+ and Cl channels (Shennan & Peaker, 2000). In the pancreas, regulation of cellular secretion of insulin could result from changes in the ionic permeability, which modulates membrane potential (Newgard & McGarry, 1995). This perspective led Silanikove et al. (2000) to test the possibility that ion channels expressed in the apical regions of mammary gland epithelium are involved in the regulation of milk secretion. Indeed, Silanikove et al. (2000) showed that a distinct plasmin-induced b-CN peptide f (1–28) is a potent blocker of K+ channels in the apical membrane of mammary epithelial cells. The production of b-CN f (1–28) by plasmin during the storage of milk in the udder represents between 8% and 12% of the total proteose–peptone fraction in whey, and matches the formation of g1-casein (the C-terminal residues 29–209) from b-CN (Andrews, 1978). b-CN f (1–28) is resistant to further degradation by plasmin (Andrews, 1978, 1983). As far as we are aware, no genetic substitutions in this part of b-CN have been found, so that all genetic variants of b-CN will result in the same fragment. All these characteristics make b-CN f (1–28) an Anti-Lactogenic hormones: e.g. Cortisol, Estrogen Lactogenic hormones: e.g. GH, Prolactin Blood side PA =plasminogen activator PAI = PA inhibitor PLG = plasminogen PL = plasmin PLI = PL inhibitor CN = casein β-CN f (1-28) =fraction 1-28 of β-CN IGFBP = IGF binding protein PAI IGFBP β-CN f (1-28) PA β-CN Gland lumen PLG PL PLI Alveoli, the basic milk secreting unit Milk stasis, or bacterial invasion Frequent milking or suckling External effects: milking, suckling, bacterial invasion Fig. 2. Overview of the PA–plasminogen–plasmin negative feedback mechanism that down-regulate milk secretion. The contribution of most elements is described in the text. Bold arrows indicate ﬂow signal along the feedback loop, dotted arrows positive effects and dashed arrows suppressive effects. ARTICLE IN PRESS N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545 ideal candidate for negative feedback control of milk secretion. Infusion of a solution composed of a casein digest enriched with b-CN f (1–28) into the cistern of cows, or infusion of pure b-CN f (1–28) into the cistern of goats, led to a transient reduction in milk secretion in the treated gland (Silanikove et al., 2000). Stress and stress-related hormones such as glucocorticoids inhibit lactation in cows (Shamay, Shapiro, Barash, Bruckental, & Silanikove, 2000). Silanikove et al. (2000) proposed a novel mechanism connecting stress with the PA–plasminogen–plasmin system. They showed that stress activates the PA–plasminogen–plasmin system leading to an increase in plasmin activity and to the formation of b-CN f (1–28). The reduction in milk production due to dehydration stress or glucocorticoid (dexamethsone) was correlated with the activity of plasmin and channelblocking activity in the milk of the tested cows (Silanikove et al., 2000). Complexes of both types of PA with PAinhibitor-1 have been detected in the culture medium of bovine mammary epithelial cells (Heegaard, White, Zavizion, Turner, & Politis, 1994). So far, a better understanding of the role of the hypothalamus–pituitary–adrenocortical axis and PA–plasminogen–plasmin system in milk has been hampered by a lack of basic knowledge of the physiological function of these two systems in milk synthesis and secretion. The results and concept presented in Fig. 2 provides an explanation for the already-known correlation between the activity of the above systems and reduced milk secretion. Accordingly, activation of the hypothalamus– pituitary–adrenocortical axis by external stress liberates cortisol into blood plasma, which in turn induces the liberation of PA from the mammary epithelial cells into the mammary cistern, where it activates the plasmin system and enhances the release of b-CN f (1–28) from b-CN. Inhibition of ion channels by b-CN f (1–28) triggers an as yet unknown process which reduces the secretion of lactose and monovalent ions into the lumen of the gland, leading to the decrease in milk volume. This rapid modulation of milk secretion increases the potential for survival in response to stress. Based on the evidence described above, this system may explain also the effects of frequency and efﬁciency of udder emptying and, as will be described below, the response in levels of milk secretion to the presence of bacterial infection of the udder. A direct association between decreased milking frequency and an increase in the activity of the PA–plasminogen–plasmin system has been found (Kelly, Reid, Joyce, Meaney, & Foley, 1998; Stelwagen, Politis et al., 1994). 2.3. Role of the plasmin system in induction of involution Mammary gland involution proceeds through several distinct stages that involve cessation of milking, apoptosis of epithelial cells and tissue remodelling. Unilateral cessation of milking in goats (Quarrie, Addey, & Wilde, 539 1994) and teat sealing in mice (Li et al., 1997; Marti, Feng, Altermatt, & Jaggi, 1997; Quarrie, Addey, & Wilde, 1998) induced involution in the treated gland only. This speciﬁcity suggests that mammary involution is triggered by local stimuli. Reinitiating milk removal can reverse the ﬁrst stage of involution, but the second stage of involution is irreversible and is characterized by activation of proteases that destroy the lobular–alveolar structure of the gland by degrading the extracellular matrix and basement membrane, and cause massive loss of alveolar cells (Capuco & Akers, 1999; Hurley, 1989). In cows, involution is complete by 21–30 days after drying-off, and during this period the mammary secretion becomes scant, watery, turbid (serum-like) and rich in leukocytes (41 106mL1) (Capuco & Akers, 1999). The compositional changes include a dramatic decrease in the concentrations of lactose and fat, and parallel increases in the concentrations of lactoferrin and immunoglobulins, which are part of the innate immune system (Capuco & Akers, 1999; Shamay, Shapiro, Leitner, & Silanikove, 2003). It takes dairy cows considerably more time than rodents to reach involution (Capuco & Akers, 1999). In cows reaching involution while still producing a lot of milk (X20–30 L day1), following cessation of milking, plasmin activity in mammary secretion increased gradually and became substantially higher within 13 days (Shamay et al., 2003). In mice, plasmin activity rose sharply immediately following the induction of drying-off (Ossowski, Biggel, & Reich, 1979), which may explain the differences between the species in their rate of involution. In support of this hypothesis, Shamay, Shapiro, Mabjeesh, and Silanikove (2002) for goats and Shamay et al. (2003) for cows, have shown that the infusion of casein hydrolyzates (CNHs) which contain products of plasmin activity dramatically accelerated the rate of involution, to the extent that it was complete within 3 days. The infusion of CNH was followed by rapid (within 3 days) drying-off of mammary secretion and was associated with earlier increases in the concentrations of components of the innate immune system: lactoferrin (an antimicrobial protein), immunoglobulin type G (Shamay et al., 2003) and the formation of free radicals with bactericidal activity by milk enzymes (Silanikove, Shapiro, Shamay, & Leitner, 2005). In conventional drying-off, induced by thee abrupt cessation of milking, ﬂuid volume declined precipitously only after 3–7 days (Hurley, 1989; Noble & Hurley, 1999; Shamay et al., 2003). It was concluded that CNH treatment induced involution in the treated gland that imitates the natural involution process, albeit at a rate which accelerates and synchronizes the involution process among the treated cows in comparison to involution induced by the cessation of milking (Shamay et al., 2002, 2003). Milk stasis also induces the disruption of tight junctions between epithelial cells due to the accumulation of local negative feedback signals (Nguyen & Neville, 1998). In CNH-treated glands, disruption of the tight junctions occurred within 8 h after the ﬁrst treatment. In comparison, ARTICLE IN PRESS 540 N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545 it took 18 h in dairy cows to ﬁnd the ﬁrst signs of tight junction leakiness (Stelwagen, Farr, McFadden, Prosser, & Davis, 1997) and 3 days to complete the process (Shamay et al., 2003). On the resumption of milking, the gland rapidly resumes tight junction integrity (Stelwagen et al., 1997). Thus, milking frequency affects the leakiness of the tight junctions, and the increased leakiness, induces reduced milk yield, increased leukocytes content in the gland and increased proteolysis of casein (Stelwagen, Politis et al., 1994; Stelwagen, Davis, Farr, Eichler, & Politis, 1994), and hence the deterioration of milk quality for the dairy industry (Kelly et al., 1998). These responses are accentuated in late lactation when there is natural increase in the activity of the PA–plasminogen–plasmin system (Kelly et al., 1998; Lacy-Hulbert, Woolford, Nicholas, Prosser, & Stelwagen, 1999). Sørensen, Muir, and Knight (2001) concluded that thrice-daily milking, in comparison to twice-daily milking, helps to prevent the usual increase in casein degradation associated with late lactation. Part of this effect is due simply to reduced exposure to plasmin as a result of the reduced storage time in the udder, but it is partly due to a better maintenance of epithelial tight junction integrity as lactation advances. Thus, the results of Sørensen et al. (2001) are in agreement with the ﬁnding that casein degradation products induce the disruption of tight junction integrity (Shamay et al., 2002, 2003). The data of Shamay et al. (2002, 2003) indicate that the ﬁrst step in the induction of involution in the mammary gland is disruption of the tight junctions between epithelial cells. Maintaining the tight junctions open for a critical time (about 48 h) initiates the second phase of involution, which is irreversible. Wilde, Blatchford, Knight, and Peaker (1989) presented data indicating that incomplete milking over a long period caused partial secretory cellular involution via a local chemical feedback mechanism in the gland. Thus, collectively, the data of Kelly et al. (1998), Lacy-Hulbert et al. (1999) and Sørensen et al. (2001) strongly suggest that the mammary gland may undergo partial involution while still in the lactogenesis phase. 2.4. The plasminogen activator–plasminogen–plasmin-based negative feedback mechanism as a working hypothesis The PA–plasminogen–plasmin-based negative feedback mechanism which down-regulates milk secretion was presented in Section 2.2 and, in Section 2.3, the PA–plasminogen–plasmin-based negative feedback mechanism which induces the disruption of tight junctions and involution was described. It was also noted that partial involution of the mammary gland may be induced even during lactogenesis. We are aware that formal approval of these two concepts should wait for further elucidation of the interaction between the casein derived peptide/s (ligand/s) and respective receptor/s or ion channel/s on the apical membrane of the secretory cells. Nevertheless, the utility of a concept can also be judged by its ability to explain current data and to provide tools that predict experimental results. In this section, we will demonstrate how these two concepts explain some apparent contradictions in respect to changes in milk composition associated with the effects of milking frequency and subclinical mastitis. Firstly, it should be emphasized that the down-regulation of milk secretion is associated with mild activation of plasmin (10–40% increase in activity). Under such conditions, the response induced by b-CN f (1–28) affects speciﬁcally ﬂuid secretion (water, lactose and ions), without affecting lactose concentration or fat and protein secretion and without affecting the integrity of tight junctions and the leukocyte level in the gland. Consequently, mild activation of the plasmin system results in reduced milk yield but with higher concentrations of fat and proteins, as seen in the response to injecting cows with dexamethasone (Shamay et al., 2000). On the other hand, massive activation of the plasmin activity (X150%) is associated with extensive degradation of casein, disruption of tight junctions, an inﬂammatory response (increase in leukocytes and NAGase activity), and a reduction in lactose concentration. As a result, ﬂuid, fat and protein secretion are negatively affected, as seen in the response to treatment with CNH of goats (Shamay et al., 2002) and cows (Shamay et al., 2003). Remond, Pomies, Dupont, and Chilliard (2004) found that once-daily milking, in comparison with twice-daily milking, reduced milk yield by 30%. The lactose concentration and SCC were similar, but fat and protein content were higher for once-daily milking. A response of a similar nature with respect to gross milk composition was reported by Smith, Ely, Graves, and Gilson (2002), who compared the effect of thrice-daily milkings with twice-daily milking in cows and Bar-Peled et al. (1995) who compared six daily milkings and 3 daily milkings plus 3 daily sucklings with thrice-daily milked cows. Similar results were also observed in twice-daily milked dairy goats in comparison with oncedaily milking (Salama et al., 2003). It seems that increased milking frequency dilute the content of b-CN f (1–28), which in turn reduces the inhibition on ﬂuid secretion. However, because b-CN f (1–28) affects fat and protein secretion less than ﬂuid secretion, it results in increasing fat and protein concentration in the milk. In contrast, when plasmin activity is already high (Kelly et al., 1998; Stelwagen, Politis et al., 1994), or when milk stasis extends much more than 12 h (Stelwagen et al., 1997), caseinderived active components disrupt the epithelial tight junctions, induces inﬂammatory response and affects ﬂuid, fat and protein secretion. Our group recently compared the effect of subclinical infection in one gland in comparison to an uninfected gland in the same animal on milk composition and yield in sheep (Leitner, Chaffer et al., 2004), goats (Leitner et al., 2004) and on milk composition in cows (Leitner, Krifucks, Merin, Lavi, & Silanikove, 2005). Milk yield was reduced in the infected glands in comparison with the uninfected ARTICLE IN PRESS N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545 ones, and these changes were associated with increases in plasmin activity, indices of inﬂammation (SCC, NAGase activity), proteolysis of casein (increased proteose–peptone content and lower casein number) and a decrease in lactose concentration. However, the results for fat, total protein and casein concentration were variable in the case of sheep, a signiﬁcant decrease in fat, total protein and casein concentration and a signiﬁcant increase in whey protein concentration were found. In the case of goats, subclinical mastitis was associated with no change in fat concentration and a signiﬁcant increase in total protein, but no change in casein level and with a signiﬁcant increase in whey protein concentration. In the case of cows, infection was not associated with changes in fat, total protein or casein, but was associated with increase of total whey protein concentration. In view of the evidence for extensive degradation of casein in the infected glands, the lack of response in total casein concentration in goats and cows is striking. This contradiction may be explained as an outcome of the reduction in casein secretion and casein content due to enhanced hydrolysis and the increased casein concentration as a result of a greater reduction in ﬂuid secretion. The more severe response in sheep, in a comparison to goats and cows, is consistent with a higher basal level of plasmin activity, a higher level of plasmin activity in the infected glands, a higher basal level of casein, and hence a higher level of casein degradation products (i.e., proteose–peptones) in the infected glands. These inter-species differences are also associated with a greater reduction in lactose concentration in sheep than in goats and cows, in line with the primary osmotic role of lactose in milk and its effect on ﬂuid secretion (Shamay et al., 2000). The fact that all glands treated with CNH (so far, 4than 100 cases in dairy cows) resumed normal lactation after parturition supports the conclusion that the treatment imitated natural phenomena rather than inducing a necrotic response which would irreversibly damage the secretory function of the udder (Shamay et al., 2003). In several aspects, such as induction of inﬂammation and changes in milk composition, CNH treatment resembles studies in which the mammary gland was challenged with endotoxin or colchicines (Olivier & Smith, 1983; Persson, Carlsson, Hambleton, & Guidry, 1992). However, this treatment cannot induce complete drying-off of mammary secretion, and the mammary gland becomes partially refractory to frequent treatment with endotoxin (Shuster & Harmon, 1991). These qualities of CNH treatment have led us to examine its use for drying-off secretion in infected mammary quarters, and evaluate its ability to cure microbiological infection. CNH treatment appears to be an effective tool for improving milk hygiene (i.e., reducing SCC) in cows exposed to sub-clinical or chronic mastitis and for eliminating bacterial infection (Silanikove, Iscovich, & Leitner, 2005). The latter effects of CNH treatment also suggest that it may be use as an effective non-antibiotic dry cow treatment. 541 2.5. Milk enzymes as part of the innate immune system Milk and colostrum contain several antimicrobial factors, which exert both speciﬁc and non-speciﬁc bacteriostatic and bactericidal activity. These factors may be used to protect the mammary gland itself or transferred from the mother to the neonate and contribute to the protection of the off-spring against infectious diseases. For many species, the milk-derived immunoglobulins are crucial for survival of the newborn. Human milk contains high level of lysozyme, which has been found to be effective at signiﬁcantly slowing the growth of, or killing various types of bacteria (e.g., Maga, Anderson, Cullor, Smith, & Murray, 1998; Reddy, Bhaskaram, Raghuramulu, & Jagadeesan, 1977). NAGase activity, which is constitutively present in human milk at a high concentration, and at a high concentration in cows’ milk during inﬂammation, also confers antimicrobial activity (Pompei, Ingianni, Cagetti, Rrizzo, & Cotti, 1993). Xanthine oxidoreductase and lactoperoxidase are two ubiquitously expressed enzymes in bovine milk which are capable of forming free radicals. The former uses xanthine, hypoxanthine, or reduced nicotinamide adenine nucleotide as electron-donating substrates and catalyzes the last two steps in the formation of urate. The enzyme is synthesized as xanthine dehydrogenase, but can be readily converted to xanthine oxidase by oxidation of sulfhydryl residues or by proteolysis (Harrison, 2002). The weak microbicidal activity of xanthine oxidase relates to its ability to form superoxide and hydrogen peroxide (Bjorck & Claesson, 1979). However, recent studies have shown that the microbicidal activity of XO in milk may also be related to the formation of NO from nitrite (Hancock et al., 2002; Li, Samouilov, Liu & Zweier, 2003; Stevens et al., 2000); XO converts nitrate into nitrite, and so may substantially increase the substrate for NO generation (Li et al., 2003). Lactoperoxidase, a heme-containing peroxidase, is likely to contribute to oxidative mechanisms in milk (Østdal, Bjerrum, Pedersen, & Andersen, 2000). Thiocyanate, considered to be the physiological substrate of LPO, is oxidized by the enzyme to hypothiocyanite, with H2O2 serving as the electron acceptor (Wolfson & Sumner, 1993). However, the lactoperoxidase/SCN/H2O2 system in milk is inactive (Althaus, Molina, Rodriguez, & Fernandez, 2001; Silanikove, Shapiro et al., 2005), and its activation requires the addition of exogenous thiocyanate and H2O2 to levels exceeding their physiological concentration in milk (Wolfson & Sumner, 1993). On the other hand, nitrite reacts with mammalian peroxidases such as lactoperoxidase to produce the potent radical, nitric dioxide (NO2 ) (Van der Vliet, Eiserich, Ilalliwell, & Cross, 1997) and this system has been shown to be active in milk (Silanikove, Shapiro et al., 2005). Silanikove, Shapiro et al. (2005) have shown that NO and H2O2 were constantly produced in the mammary gland secretions. Nitrite formed either by autooxidation of NO or by conversion of nitrate to nitrite by xanthine oxidase ARTICLE IN PRESS 542 N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545 was converted into the powerful nitric dioxide radical by lactoperoxidase and H2O2 which is derived from the metabolism of xanthine by xanthine oxidase. Nitric dioxide is most likely responsible for the formation of nitrosothiols on thiol-bearing groups, which allow prolonged presence of NO in mammary secretion. Nitrite is effectively converted to nitrate, which accumulated in the range of 25–1000 mM from the start of involution (i.e., in normal milk) to the complete involution of glands. The conversion of active nitrite into less active nitrate is mediated by catalase. Catalase, therefore, appears to play a central role in redox control in milk (Silanikove, Shapiro et al., 2005). The secretion of all mammary glands was bactericidal and bacteriostatic during established involution, and this appeared sooner and more acutely in glands treated with CNH, within 8–24 h. It was concluded that xanthine oxidase, lactoperoxidase and NO are components of the mammary innate immune system that has bactericidal and bacteriostatic activity in mammary secretions. The innate immune system plays a major role in preventing intramammary infection during milk stasis and its activation may increase its effectiveness. In other species, other enzymes that produce radicals may be important for gland defense; for example, in mouse milk, H2O2 produced using endogenous amino acids by L-amino acid oxidase kills bacteria in the mammary gland (Sun et al., 2002). 3. Conclusion Milk enzymes have an important biological role and so far have been found to be involved in the control of milk secretion, developmental stage (involution), the gland innate immune system and preventing oxidative damage to its essential nutrients. Many more secrets are probably waiting to be revealed. 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