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Hormones in bovine milk and milk products: A survey
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International Dairy Journal 16 (2006) 1408–1414 www.elsevier.com/locate/idairyj
Review
Hormones in bovine milk and milk products: A survey Pierre-Nicolas Jouana,, Yves Pouliotb, Sylvie F. Gauthiera, Jean-Paul Laforestc a
Faculte´ de Me´decine et de Pharmacie, Universite´ de Rennes I, France Groupe STELA, Institut des Nutraceutiques et Aliments Fonctionnels (INAF), Universite´ Laval, Que´bec., Canada G1K 7P4 c De´partement des Sciences Animales, Faculte´ des Sciences de l’Agriculture et de l’Alimentation, Universite´ Laval, Que´bec., Canada G1K 7P4 b
Received 9 September 2005; accepted 31 May 2006
Abstract A survey of the published data on the occurrence of hormones in milk and milk products is presented. Bovine milk and colostrum contain a large number of hormones from either steroidic or peptidic origin. The main categories to which these molecules belong are gonadal (estrogens, progesterone, androgens), adrenal (glucocorticoids), pituitary (prolactin, growth hormone) and hypothalamic hormones (GRH, LH-RH, TRH). Other molecules, such as proteins related to the parathyroid hormone, insulin, somatostatin, calcitonin, bombesin, erythropoeitin and melatonin, are also of interest. The exact role of hormones in the development of the newborn is still not known, but it is believed that they may contribute to the growth of the newborn and to the development and maturation of its gastrointestinal and immune systems. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bovine milk; Milk products; Colostrum; Hormones; Peptides
Contents 1. 2.
3. 4.
5.
6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gonadal hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Progesterone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Androgens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adrenal gland hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pituitary hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Prolactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Growth hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypothalamic hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Gonadotropin-releasing hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Luteinizing hormone-releasing hormone (LH-RH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Thyrotropin-releasing hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Somatostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Parathyroid hormone-related protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Calcitonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Bombesin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +418 656 5988; fax: +418 656 3353.
E-mail address: [email protected] (P.-N. Jouan). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.06.007
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6.5. Erythropoietin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Melatonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Yaida (1929) and Ratsimamanga, Nigeon-Dureuil, and Rabinowiez (1956) were the first authors to report hormones in bovine milk, essentially steroidic hormones of gonadal and adrenal origins, respectively. Since these early findings, a large number of studies, mostly published in the late 1970s and 1980s, have been devoted to the finding and dosage of hormones in cows’ milk. However, because hormones in milk have very little nutritive or diagnostic value, scientific literature on their concentrations in milk has not progressed. Hormonal data are mainly associated with physiological studies in which hormonal concentrations have been determined in the blood. Hormones in human and bovine milk have been reviewed by Koldovsky and Thornburg (1987), and Grosvenor, Picciano, and Baumrucker (1993). Hormones found in milk originate from the blood flow and are secreted in milk through an active transport within the mammary gland. Some hormones can also be synthesized Table 1 Summary of the main hormones detected in bovine milk Hormone
Ranges reported in bovine milka
Gonadal hormones Estrogens Progesterone Androgens
5–10 pg mL–1 2–20 ng mL–1 0–50 pg mL–1
Adrenal gland hormones Glucocorticoids (cortisosterone, cortisol) 5a-androstene-3,17-dione
0–50 ng mL–1 3 ng mL–1
Pituitary hormones Prolactin Growth hormone (GH)
5–200 ng mL–1 0–1 ng mL–1
Hypothalamic hormones Gonadotropin-releasing hormone (GRH) Luteinizing hormone-releasing hormone (LHRH) Thyrotropin-releasing hormone (TRH) Somatostatin Other hormones Parathyroid hormone-related protein (PH-rP) Insulin Calcitonin Bombesin (gastrin-releasing peptide) Erythropoietin Melatonin a
0.5–3.0 ng mL–1 0.5–3.0 ng mL–1 0–0.5 ng mL–1 10–30 ng mL–1 40–100 ng mL–1 5–40 ng mL–1 700 ng mL–1 0.25–450 ng mL–1 n/ab 5–25 pg mL 1
See text for details. Occurrence in milk suspected but no analytical data available.
b
by the mammary gland and excreted to milk. Their content in the mammary gland and in milk often exceeds that in the maternal blood plasma. This indicates that hormones in maternal milk may be involved, in situ, in the regulation of specific functions of the mammary gland, and they may also contribute to the growth of the newborn and to the development and maturation of its gastrointestinal tract and immune systems. Hormones found in milk could also regulate, at least temporarily, the activity of some endocrine glands until the newborn’s hormonal system reaches maturity (Bernt & Walker, 1999). Hormones already reported in milk are summarized in Table 1. The main categories to which these molecules belong are gonadal, adrenal, pituitary and hypothalamic hormones. Other molecules, such as proteins related to the parathyroid hormone, insulin, somatostatin, calcitonin, bombesin, erythropoietin and melatonin, also have been reported, and are discussed in this paper. The various ranges of concentrations for these hormones in bovine milk are included in the Table 1 for comparison purposes, but will be discussed throughout the present paper.
2. Gonadal hormones 2.1. Estrogens A number of techniques have been used to quantify estrogen content in milk, including colorimetry, spectrofluorometry, gas chromatography and high-pressure chromatography. However, the most reliable data were obtained using radioimmunoassay (RIA). This was used by Wolford and Argoudelis (1979) to estimate 17bestradiol (E2), estrone (E1) and estriol (E3) concentrations in milk and in a number of dairy products. Concentrations of E2 were found to be 10.3–14.4 pg mL–1 in raw milk and 5.071.2–9.079 pg mL–1 in skimmed milk. Concentrations of E1 were found to be 5.5–8.0 pg mL–1 in raw milk and 9.171.2–20.271.7 pg mL–1 in skimmed milk. These data show that approximately 65% of E2 and 80% of E1 can be found in the milk fat fraction. The occurrence of estrogens in both butter and skim milk clearly indicates a repartition of those steroids between the lipid and serum phases of milk. E1 is the predominant estrogen in milk. Its content is markedly higher than that of E2. In whey, 48% of E2 and 53% of E1 are bound to proteins. It is likely that these hormones are associated with bovine serum albumin since, in blood flow, these estrogens are transported by plasmatic serum albumin.
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Estrogens concentrations were also estimated using RIA by Monk, Erb, and Mollet (1975) during the gestation and the cycle. Before estrus, the average E2 content was 25.876 pg mL–1, increasing to 84741 pg mL–1 at estrus and decreasing to 42.4711.2 pg mL–1 after estrus. Average contents of E1 were 34.6679.8, 5876 and 5175.6 pg mL–1 during proestrus, estrus and metestrus, respectively. Concentrations of E2 and E1 found in milk were higher than those found in blood plasma throughout the ovarian cycle. They typically average 37.676 pg mL–1 for E2 and 4372 pg mL–1 for E1 in milk, whereas they average 1672 pg mL–1 for E2 and 1071 pg mL–1 for E1 in blood. Monk et al. (1975) also determined E2 and E1 contents in milk at three stages of gestation. The authors did not report any increase in estrogens during the cycle, but noticed an increase in plasmatic contents of E2 but not E1, contrary to what has been reported during the ovarian cycle. Estrogen concentrations are generally higher in milk than in the blood flow. This observation suggests the occurrence of an uptake of estrogens by the mammary gland. It also suggests that, since E1 content is higher than E2, the mammary gland either preferentially concentrates estrone or it might be able to convert, in situ, part of estradiol into estrone. Erb, Chew, and Keller (1977) have used RIA to determine the estrogen content in plasma and milk during the pre-partum period and lactation. During lactation (days 3–25), E2 concentrations were 2172 pg mL–1 in plasma and 13 pg mL–1 in milk. Contents of E1 were 5872 pg mL–1 in plasma and 2873 pg mL–1 in milk. These authors detected the 17a isomer (17a-estradiol), at a higher concentration in milk than in plasma (160714 and 15 pg mL–1, respectively). During the pre-partum period (days 3 to 1), E1, E2 and the epimeric form E2a represented 66%, 19% and 15% of total estrogens, respectively. Therefore, E1 is the predominant estrogen in milk. A similar pattern was found for plasma with E1, E2 and E2a representing 57%, 19% and 24% of total estrogens, respectively. During lactation (days 0–2), proportions of total estrogens represented by E1, E2 and E2a were, respectively, 55%, 19% and 26% in milk and 53%, 22% and 25% in plasma. During days 3–25 of lactation, E2 represented 7%, E2a 79% and E1 14% of total estrogens in milk, compared with 22%, 16% and 62%, respectively, in plasma. These data show that E2 contents are stable during the pre-partum period and lactation. On the contrary, E2a concentrations increased during lactation, whereas that of E1, which were high during the pre-partum period and first days of lactation (days 0–2), decreased thereafter (days 3–25 of lactation).
Harkness, 1974). This hormone was absent from colostrum. Its presence has been reported in milk approximately 15 days after parturition. Between days 15 and 57 of lactation, Pg concentrations in milk varied in a cyclic manner between 1.2 and 5.9 ng mL–1. Pg concentrations were higher in milk collected in the evening than in that collected in the morning. In addition to Pg, these authors have also reported the occurrence of 5a-pregnane-3,20dione, a metabolite of Pg, in many blood samples from pregnant cows. Milk Pg determination is now used for pregnancy diagnosis in cows (Comin et al., 2005). Pg is found in milk at concentrations of approximately 0.3–0.4 pg mL–1. Darling et al. (1974) also found that Pg concentrations are higher in cream than in skim milk. As for estrogens, Pg concentrations were found to be higher in milk than in blood plasma (Heap, Gwyn, Laing, & Walters, 1973). Ginther, Nuti, Wentworth, and Tyler (1974) have used RIA to determine Pg concentrations in cow milk during pregnancy on days 30, 60, 90, 120, 150, 180 and 210. It varied between 15.1 and 26.2 ng mL–1. These concentrations were generally four times higher than those in blood plasma (on average 21.2 ng mL–1 in milk compared with 5.3 ng mL–1 in plasma). Pg content in milk exceeds that of plasma already 4–5 days before and after calving (Erb et al., 1977). After calving, Pg content of milk decreases rapidly but remains higher than that of plasma and follows plasmatic variations of Pg. Pg contents have been determined in a number of dairy products (Ginther, Nuti, Garcia, Wentworth, & Tyler, 1976; Hoffmann, Hamburger, & Karg, 1975). Both studies reported similar data. According to Ginther et al. (1976), whole milk, skim milk and cream contain 11.376, 4.674 and 58.775.3 ng mL–1 of Pg, respectively. As for the commercial products, concentrations were 9.575 ng mL–1 in whole milk, 2.176 ng mg–1 in skim milk, 72.775.8 ng mL–1 in cream and 132.975.1 ng mL–1 in butter. The values were 98 ng mL–1 in whole milk powder and 17 ng g–1 in skim milk powder (Hoffmann et al., 1975). The only discrepancy reported concerned butter for which a value of 132.975.1 ng g–1 in Pg was reported by Ginther et al. (1976) and 300 ng g–1 by Hoffmann et al. (1975).
2.2. Progesterone
3. Adrenal gland hormones
Progesterone (Pg) concentrations have been determined first in bovine plasma by gas chromatography during pregnancy and after parturition (Darling, Laing, &
The occurrence of corticosteroids in cows’ milk has been demonstrated for the first time in the late 1950s (Ratsimamanga et al., 1956). According to Gwazdauskas, Pappe, &
2.3. Androgens Very few studies have been published on androgen content of milk. Testosterone in milk has been determined by RIA after ether extraction and purification on a silica gel. The data reported varied from non-detectable to 50 pg mL–1 in milk at estrus and 150 pg mL–1 during the luteal phase. The ratio between free and conjugated testosterone is 1/1 (Hoffman & Rattenberger, 1977).
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Mc Gilliard, (1977), glucocorticoid concentrations in milk vary between 0.7 and 1.4 ng mL–1. There is no difference between whole and skim milk. Corticosteroı¨ d contents of milk are much lower than those of blood plasma. According to Tucker and Schwalm (1977) milk cortisol represents only 10–23% and corticosterone 60–90% of their plasmatic homologs. Injections of ACTH to cows cause a temporary increase in milk glucocorticoids. Similarly, administration of hydrocortisone generates an increase of glucocorticoids in milk (Gwazdauskas et al., 1977). Cortisol and corticosterone are the main glucocorticoids in blood plasma of cows. Their detection has been achieved by a radio-binding assay after extraction from the milk using ethyl acetate and chromatographic separation using Sephadex LH-20 (Tucker & Schwalm, 1977). During lactation (0–11 months), glucocorticoid concentrations in milk only represent 4% of the blood plasma concentrations, indicating that only a small amount is transferred to the milk. However, the relative proportions of the different glucocorticoid hormones differ between blood plasma and milkrent. For instance, cortisol is predominant in blood plasma (66% of total glucocorticoids) whereas corticosterone represents 90% of total glucocorticoids in milk. During lactation, glucocorticoid concentrations in milk decrease gradually and significantly. They go from 0.5970.11 ng mL–1 during the first 2 months, to 0.287 0.04 ng mL–1 between 5–7 months and 0.2570.02 ng mL–1 between 9 and 11 months of lactation. This decrease is essentially related to changes in cortisol concentrations, while corticosterone concentrations slightly increase during the same period. Contrary to estrogens, glucocorticoids are not concentrated in cream. Indeed, 71–89% of the milk estrogens will be found In the cream fraction, whereas it contains only 12–15% of the corticosteroids. In cows’ milk, corticosteroids are equally distributed between caseins and whey protein fractions. Gorewit and Tucker (1976, 1977), studying the binding of glucocorticoids in the mammary gland of lactating cows, have demonstrated the presence of glucocorticoid receptors. Sections of mammary glands were incubated for 1 h, at 37 1C, in the presence of cortisol-3H or hexamethasone-3H. The repartition of the compound between supernatant and pellet of a 700 g centrifugate showed the high affinity of glucocorticoid receptors in the mammary gland of the lactating cow. It was estimated that the binding constant (Kd) with cortisol was between 10–8 and 10–10 M. Also, it has been estimated that each mammary cell contained approximately 7500 high-affinity binding sites (Tucker & Schwalm, 1977). Glucocorticoids possibly act in conjunction with other hormones to maintain lactation. Their effects would be mediated by specific receptors. Glucocorticoids also seem to reduce, in a dose-dependent manner, glucose uptake by the mammary gland. Glucose is an important substrate for lactose production. Lactose content may therefore be
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regulated by glucocorticoids, which explains the decrease of milk production when glucocorticoids content is high. The steroid 5a-androstane-3,17-dione originates from the reduction of androstenedione following the action of 5a-oxydo-reductase, in the surrenal glands. Darling et al. (1974) achieved its isolation in milk by gas chromatography. However, it was only found in 3 out of 12 colostrum samples. This steroid appears in milk right after calving. Between days 4 and 57 of lactation, the average content of androstanedione in milk is 3 ng mL–1 whereas during pregnancy (3–136 days), it is 270.09 ng mL–1 of milk. This concentration remains constant throughout pregnancy. Higher concentrations of this hormone are found during the evening compared to the morning milking. 4. Pituitary hormones 4.1. Prolactin Prolactin has been detected first in cows’ milk using RIA with a double antibody (Malven & Mc Murtry, 1974), with contents varying from 5 to 200 ng mL–1. However, the numerous milk samples analyzed by the authors allowed the determination of an average value at 50 ng mL–1. Gala, Forsyth, and Turvey (1980) have determined the prolactin content of five milk samples using RIA after purification on a Sephadex G100 column. This column pre-purification allowed the recovery of most of the prolactin in a homogeneous peak. Prolactin content varied between 3.7 and 14.2 ng mL–1. The bioactivity of milk prolactin has been demonstrated in vitro on explants of mammary glands from pseudopregnant rabbits (Gala et al., 1980). Prolactin concentrations appear to fluctuate seasonally and are higher in summer than in winter. Storage of milk at 4 1C does not affect prolactin concentrations whereas 59% is lost upon storage at 15 1C (Malven & Mc Murtry, 1974). Part of the prolactin content of milk is associated with milk fat globules since a centrifugal separation of fat removes also 60% of the initial prolactin content of milk (Malven & Mc Murtry, 1974). The prolactin content of colostrum is much higher than that of milk, varying between 500 and 800 ng mL–1, compared to 6–8 ng mL–1 in milk (Kacsoh et al., 1991). Prolactin in milk probably originates from blood plasma. Its biological functions are not clearly established. It could stimulate lactation by a direct action on secretory cells. In the newborn, the permeability of the gastro-intestinal tract allows the passage of prolactin from the intestine to the blood. In the adult, prolactin is probably hydrolyzed in the intestine. 4.2. Growth hormone The growth hormone (GH) or somatotropin, in milk was first detected using RIA (Torkelson, 1987) at concentrations lower than 1 ng mL–1. Treatment of early lactating
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cows with bovine somatotropin (bST) does not induce significant increases in the milk concentration of this hormone (de Boer, Robinson, & Kennelly, 1991). GH is thought to be acting on the mammary gland by means of specific receptors as shown by Glimm, Baracos, and Kennelly (1988). It was also found that GH increased the concentration of insulin-like growth factor-1 (IGF-I) in epithelial cells of the mammary gland of lactating cows (Glimm et al., 1988). 5. Hypothalamic hormones 5.1. Gonadotropin-releasing hormones Gonadotropin-releasing hormone (GnRH) was detected and quantified in cows’ milk by Baram, Koch, Hazum, and Frikin (1977). After acid precipitation of caseins from reconstituted skim milk powder, GnRH was extracted by an acid-methanol procedure and analyzed by RIA. Concentrations of GnRH in milk vary between 0.5 and 3 ng mL–1. GnRH extracted from milk has similar biological activities as the hypothalamic hormone since it induces the release of LH and FSH from rat pituitary glands incubated in vitro. GnRH content in milk is 5–6 times greater than in blood plasma. GnRH might be from an extra-hypothalamic origin but it is more likely coming from an active transport by the mammary gland. In the newborn, GnRH is absorbed by the intestine in an active form. It may be involved in the masculinization of the male hypothalamus by stimulating androgens secretion that would act on the brain. Gonadotropin-releasing hormone-associated peptide (GAP) was also found in bovine colostrum, using RIA (Zhang et al., 1990). This 56-amino acids peptide has a sequence identical to the C-terminal end of GnRH. GAP could therefore be the precursor of GnRH. GAP concentration in defatted colostrum is 1.570.1 pmol g–1. GAP also induces the liberation of LH and FSH from rat pituitary glands incubated in vitro. According to Zhang et al. (1990), GAP could be synthesized in the mammary gland. 5.2. Luteinizing hormone-releasing hormone (LH-RH) LH-RH content in milk and colostrum has been determined by RIA, after methanol-acid extraction and HPLC (Amarant, Fridkin, & Koch, 1982). In the colostrum, LH-RH concentration is 11.7870.72 ng mL–1, whereas in milk it varies between 0.5 and 3 ng mL–1. Milk or colostrum contents of LH-RH exceed that of blood plasma. The origin of LH-RH in milk is still unknown. This hormone may originate from the blood and be concentrated in the mammary gland by an active process or be of an extra-hypothalamic origin. LH-RH from milk is biologically active. It induces the liberation of LH from rat pituitary glands incubated in vitro. LH-RH is absorbed in intact and active form by the newborn’s intestine. Milk
could be considered as a source of LH-RH for the newborn stimulating the secretion of the pituitary gonadotropins. 5.3. Thyrotropin-releasing hormone Thyrotropin-releasing hormone (TRH) has been measured in milk and colostrum by RIA (Amarant et al., 1982), using a procedure similar to that of LH-RH. TRH concentrations in colostrum and milk are 0.1670.03 and 0.05 ng mL–1, respectively. Both colostrum and milk show higher concentrations of TRH than blood plasma. As for the other hypothalamic hormones, the origin of TRH in milk is unclear. TRH is absorbed in intact and active form by the newborn’s intestine. The intestinal hydrolysis of TRH is low during the first 3 weeks after birth. It is possible that TRH modulates TSH secretion in the newborn. 5.4. Somatostatin The occurrence of somatostatin in cows’ milk has been demonstrated by enzyme immunoassay (EIA) on defatted, decaseinated milk (Takeyama et al.,1990). Somatostatin concentrations in milk vary between 10 and 30 pmol L–1. It does not seem to be affected by parturition. 6. Other hormones 6.1. Parathyroid hormone-related protein Many authors have mentioned that parathyroid hormone-related protein (PTH-rP) is present in cows’ milk. The RIA analysis of this hormone was possible after partial purification by affinity chromatography using Sepharose gel coupled with polyclonal antibody anti-PTH 1-34. The PTH-rP content in milk has been estimated to 95.6734 ng mL–1 (Budayr et al., 1989). Ratcliffe et al. (1990) obtained similar results on both fresh and pasteurized milk (86.8720.8 ng mL 1 and 73.377.1 ng mL–1), respectively. This suggests that PTH-rP is, to some extent, heat-stable and not affected by pasteurization. Purified extracts of PTH-rP were analyzed by SDS-PAGE and immuno-blotting. Two biologically active molecular forms (27 and 21 kDa) were detected (Ratcliffe et al., 1990). Law et al. (1991) reported PTH-rP concentrations in whole and pasteurized milk as 59.2718.5 and 66.77 12.5 ng mL–1, respectively. The breed of cow seems to have an influence on PTH-rP content, since milk from Jersey cows contained 52.675.4 ng mL–1 whereas that from Friesians cows had 41.074.8 ng mL–1 (Law et al., 1991), and this difference was statistically significant (Po0,01). PTH-rP content of milk increased gradually during lactation (Goff, Reinhardt, Lee, & Hollis, 1991). PTH-rP contents do not seem to vary in commercial dairy products. Budayr et al. (1989) showed that the PTH-rP content was 81.4717.9 ng mL–1 in whole milk, 88.978.7 ng mL–1 in low-fat milk, 118.3718.9 ng mL–1 in
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skim milk and 76.2732.7 ng mL–1 in reconstituted skim milk powder. The physiological functions of PTH-rP have not been clearly established. Produced by the mammary gland, this hormone might be involved in the transport of calcium from blood plasma to milk. It would therefore ensure the calcium supply to the newborn. However, this hypothesis is disputable since Goff et al. (1991) found no correlation between PTH-rP concentrations and calcium content in milk. PTH-rP also may be involved in the maturation of intestinal epithelium in intestinal motility. 6.2. Insulin Insulin content in colostrum is between 0.67 and 5.0 nM, which is 100-fold higher than the concentration in the blood plasma (Ballard, Nield, Francis, Dahlenburg, & Wallace, 1982). According to Malven (1977), insulin concentrations in milk varied from 37.1714 ng mL–1 during the pre-partum period, to 6.272.1 ng mL–1 during post-partum and 5.570.6 ng mL–1 after parturition.
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concentration (5–25 pg mL 1). Its concentration in milk shows a diurnal pattern (maximal concentration at midnight and minimal concentration at noon), which parallels that of serum to some extent. It has been suggested that night-time milk could serve as source of melatonin to improve sleep and diurnal activity in elderly people (Valtonen, Niskanen, Kangas, & Koskinen, 2005). 7. Conclusion Bovine milk and milk products contain a number of hormonal substances originating from the cow’s blood flow or synthesized in the mammary gland. Although occurrence of these hormones in milk may benefit the newborn, it is not clear whether they can bring health benefits to humans in milk products. There is a need to update the data concerning hormonal levels in milk and milk products, especially in the light of changes in the genetic background of dairy cattle in the last decades, as well as in animal feeding and husbandry and new processes that have emerged in milk industry.
6.3. Calcitonin Calcitonin concentrations in human milk has been estimated at 700 ng mL–1 using RIA (Koldovsky, 1989). Calcitonin inhibits the liberation of prolactin. 6.4. Bombesin Bombesin (gastrin-releasing peptide) is a 14 amino acids peptide that is known to influence the gastric hormonal secretions following ingestion (Lazarus, Gaunido, Wilson, & Erspamer, 1986). Satiety, blood sugar concentrations, gut acidity and concentrations of some gastro-intestinal hormones are known to be influenced by bombesin. Bombesin has been found in human milk, cows’ milk, milk powder and whey (Lazarus et al., 1986). Concentrations in human, bovine and porcine milks range from 0.25 to 450 ng mL–1 (Koldovsky, 1989). 6.5. Erythropoietin Human milk is known to contain erythropoietin (Grosvenor et al., 1993). It has been suggested that erythropoietin is being transferred from the mother to the offspring by the milk and that it might be able to stimulate erythropoiesis in the offspring (Grosvenor et al., 1993). No analytical data are available for cows’ milk. 6.6. Melatonin Melatonin is a hormone synthesized by the pineal gland in a diurnal pattern reflecting photoperiodicity. Melatonin has been found in human, bovine and goat milk (Eriksson, Valtonen, Laitinen, Paananen, & Kaikkonen, 1998; Valtonen, Kangas, Voutilainen, & Eriksson, 2003) at a low
References Amarant, T., Fridkin, M., & Koch, Y. (1982). Luteinizing-hormonereleasing hormone and thyrotropin-releasing hormone in human and bovine milk. European Journal of Biochemistry, 127, 647–650. Ballard, F., Nield, M., Francis, G., Dahlenburg, G., & Wallace, J. (1982). The relationship between the insulin content and inhibitory effects of bovine colostrum on protein break down in cultured cells. Journal of Cell Biology, 110, 249–254. Baram, T., Koch, Y., Hazum, E., & Frikin, M. (1977). Gonadotropinreleasing hormone in milk. Science, 198, 300–302. Bernt, K. M., & Walker, W. A. (1999). Human milk as carrier of biochemical messages. Acta Pediatrica, 430(Suppl.), 27–41. Budayr, A., Halloran, B., King, J., Diep, D., Nisseyson, N., & Strewler, G. (1989). High levels of parathyroid hormone-like protein in milk. Proceedings of the National Academy of Science USA, 86, 7183–7185. Comin, A., Renaville, E., Marchini, E., Maiero, S., Cairoli, F., & Prandi, A. (2005). Techincal note: Direct enzyme immunoassay of progesterone in bovine milk whey. Journal of Dairy Science, 88, 4239–4242. Darling, J., Laing, A., & Harkness, R. (1974). A survey of the steroids in cow’s milk. Journal of Endocrinology, 62, 291–297. de Boer, G., Robinson, P., & Kennelly, J. (1991). Hormonal response to bovine somatotropin and dietary protein in early lactating cows. Journal of Dairy Science, 74, 2623–2632. Erb, R., Chew, B., & Keller, H. (1977). Relative concentrations of estrogen and progesterone in milk and blood and excretion of estrogen in urine. Journal of Animal Science, 46, 617–626. Eriksson, L., Valtonen, M., Laitinen, J. T., Paananen, M., & Kaikkonen, M. (1998). Diurnal rhythm of melatonin in bovine milk: Pharmacokinetics of exogeneous melatonin in lactating cows and goats. Acta Veterinaria Scandinavia, 39, 310. Gala, R., Forsyth, I., & Turvey, A. (1980). Milk prolactin is biologically active. Life Sciences, 26, 987–993. Ginther, O., Nuti, L., Garcia, M., Wentworth, B., & Tyler, W. (1976). Factors affecting progesterone concentration in cow’s milk and dairy products. Journal of Animal Science, 42, 155–159. Ginther, O., Nuti, L., Wentworth, B., & Tyler, W. (1974). Progesterone concentration in milk and blood during pregnancy in cows. Proceedings of the Society for Experimental Biology and Medicine, 146, 354–357.
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Glimm, D., Baracos, V., & Kennelly, J. (1988). Effect of bovine somatotropin on the distribution of immunoreactive insulin-like growth factor-1 in lactating bovine mammary tissue. Journal of Dairy Science, 71, 2923–2935. Goff, J., Reinhardt, T., Lee, S., & Hollis, B. (1991). Parathyroid hormonerelated peptide content of bovine milk and calf blood assessed by radioimmunoassay and bioassay. Endocrinology, 129, 2815–2819. Gorewit, R. C., & Tucker, H. A. (1976). Corticoid binding in mammary tissue slices from lactating cows. Journal of Dairy Science, 59, 232–240. Gorewit, R. C., & Tucker, H. A. (1977). Lactational events related to glucocorticoid binding in bovine mammary tissue. Journal of Dairy Science, 60, 889–895. Grosvenor, C. E., Picciano, M. F., & Baumrucker, C. R. (1993). Hormones and growth factors in milk. Endocrine Reviews, 14, 710–728. Gwazdauskas, F., Pappe, M., & Mc Gilliard, M. (1977). Milk and plasma glucocorticoid alterations after injections of hydrocortisone and adrenocorticotropin. Proceedings of the Society for Experimental Biology and Medicine, 154, 543–545. Heap, R., Gwyn, M., Laing, J., & Walters, D. (1973). Pregnancy diagnosis in cows: Changes in milk progesterone, concentration during the oestrous cycle and pregnancy measured by rapid radioimmunoassay. Journal of Agricultural Sciences, 81, 151–159. Hoffmann, B., Hamburger, R., & Karg, H. (1975). Natu¨rliche Vorkommen von Progesterone in hendelsu¨blichen Milchprodukten. Zeitschrift fu¨r Lebensmittel–Untersuchung und –Forschung, 158, 253. Hoffman, B., & Rattenberger, E. (1977). Testosterone concentrations in tissue from veal calves, bulls and heifers and in milk samples. Journal of Animal Science, 46, 635–641. Kacsoh, B., Toth, B., Avery, L., Deaver, D., Baumrucker, C., & Grosvenor, C. (1991). Biological and immunological activities of glycosylated and molecular weight variants of bovine prolactin in colostrum, and milk. Journal of Animal Science, 69, 456. Koldovsky, O. (1989). Search for role of milk-borne biologically active peptides for the suckling. Journal of Nutrition, 119, 1543–1551. Koldovsky, O., & Thornburg, W. (1987). Hormones in milk. Journal of Pediatric Gastroenterology and Nutrition, 6, 172–196. Law, F., Moate, P., Leaver, D., Diefenbach-Jagger, H., Grill, V., Ho, P., et al. (1991). Parathyroid hormone-related protein in milk and its correlation with bovine milk calcium. Journal of Endocrinology, 128, 21–26. Lazarus, L. H., Gaunido, G., Wilson, W. E., & Erspamer, V. (1986). An immunoreactive peptide in milk contains bombesin-like bioactivity. Experientia, 42, 822–823.
Malven, P. (1977). Prolactin and other hormones in milk. Journal of Animal Science, 45, 609–616. Malven, P., & Mc Murtry, J. (1974). Measurement of prolactin in milk by radio immunoassay. Journal of Dairy Science, 57, 411–415. Monk, E., Erb, R., & Mollet, T. (1975). Relationship between immunoreactive estrone and estradiol in milk, blood and urine of dairy cows. Journal of Dairy Science, 58, 34–40. Ratcliffe, W., Green, E., Emly, J., Norbury, S., Lindsay, M., Heath, D., et al. (1990). Identification and partial characterization of parathyroid hormone-related protein in human and bovine milk. Journal of Endocrinology, 127, 167–176. Ratsimamanga, A., Nigeon-Dureuil, M., & Rabinowiez, M. (1956). Pre´sence d’hormone type cortinique dans le lait de la vache gravide. Compte-Rendus de la Socie´te´ de Biologie, 150, 2179–2182. Takeyama, M., Yanaga, N., Yarimizu, K., Ono, J., Takaki, R., Fujii, M., et al. (1990). Enzyme immunoassay of somatostatin (SS)-like immunoreactive substance in bovine milk. Chemical Pharmacology Bulletin, 38, 456–459. Torkelson, A. (1987). Radioimmunoassay of somatotropin in milk from cows administered recombinant bovine somatotropin. Proceedings of the American Dairy Science Association(Suppl. 1), 70 146. Tucker, H., & Schwalm, J. (1977). Glucocorticoids in mammary tissue and milk. Journal of Animal Science, 45, 627–634. Valtonen, M., Kangas, A. P., Voutilainen, M., & Eriksson, L. (2003). Diurnal rythm of melatonin in young calves and intake of melatonin in milk. Animal Science, 77, 149–154. Valtonen, M., Niskanen, L., Kangas, A. P., & Koskinen, T. (2005). Effect of melatonin-rich night-time milk on sleep and activity in elderly institutionalized subjects. Nordic Journal of Psychiatry, 59, 217–221. Wolford, S., & Argoudelis, C. (1979). Measurement of estrogen in cow’s milk, human milk and dietary products. Journal of Dairy Science, 62, 1458–1463. Yaida, N. (1929). Ovarial hormone in blood of pregnant women, of pregnant animals: Ovarial hormone in urine of pregnant women: Ovarial hormone in milk of pregnant animals. Transactions of the Japanese Pathology Society, 189, 93–101. Zhang, T., Iguchi, K., Mochizuki, T., Hoshino, M., Yanaihara, C., & Yanaihara, N. (1990). Gonadotropin-releasing hormone-associated peptide immunoreactivity in bovine colostrum. Experimental Biology and Medicine, 194, 270–273.
International Dairy Journal 16 (2006) 1408–1414 www.elsevier.com/locate/idairyj
Review
Hormones in bovine milk and milk products: A survey Pierre-Nicolas Jouana,, Yves Pouliotb, Sylvie F. Gauthiera, Jean-Paul Laforestc a
Faculte´ de Me´decine et de Pharmacie, Universite´ de Rennes I, France Groupe STELA, Institut des Nutraceutiques et Aliments Fonctionnels (INAF), Universite´ Laval, Que´bec., Canada G1K 7P4 c De´partement des Sciences Animales, Faculte´ des Sciences de l’Agriculture et de l’Alimentation, Universite´ Laval, Que´bec., Canada G1K 7P4 b
Received 9 September 2005; accepted 31 May 2006
Abstract A survey of the published data on the occurrence of hormones in milk and milk products is presented. Bovine milk and colostrum contain a large number of hormones from either steroidic or peptidic origin. The main categories to which these molecules belong are gonadal (estrogens, progesterone, androgens), adrenal (glucocorticoids), pituitary (prolactin, growth hormone) and hypothalamic hormones (GRH, LH-RH, TRH). Other molecules, such as proteins related to the parathyroid hormone, insulin, somatostatin, calcitonin, bombesin, erythropoeitin and melatonin, are also of interest. The exact role of hormones in the development of the newborn is still not known, but it is believed that they may contribute to the growth of the newborn and to the development and maturation of its gastrointestinal and immune systems. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bovine milk; Milk products; Colostrum; Hormones; Peptides
Contents 1. 2.
3. 4.
5.
6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gonadal hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Progesterone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Androgens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adrenal gland hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pituitary hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Prolactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Growth hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypothalamic hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Gonadotropin-releasing hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Luteinizing hormone-releasing hormone (LH-RH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Thyrotropin-releasing hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Somatostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Parathyroid hormone-related protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Calcitonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Bombesin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +418 656 5988; fax: +418 656 3353.
E-mail address: [email protected] (P.-N. Jouan). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.06.007
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6.5. Erythropoietin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Melatonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Yaida (1929) and Ratsimamanga, Nigeon-Dureuil, and Rabinowiez (1956) were the first authors to report hormones in bovine milk, essentially steroidic hormones of gonadal and adrenal origins, respectively. Since these early findings, a large number of studies, mostly published in the late 1970s and 1980s, have been devoted to the finding and dosage of hormones in cows’ milk. However, because hormones in milk have very little nutritive or diagnostic value, scientific literature on their concentrations in milk has not progressed. Hormonal data are mainly associated with physiological studies in which hormonal concentrations have been determined in the blood. Hormones in human and bovine milk have been reviewed by Koldovsky and Thornburg (1987), and Grosvenor, Picciano, and Baumrucker (1993). Hormones found in milk originate from the blood flow and are secreted in milk through an active transport within the mammary gland. Some hormones can also be synthesized Table 1 Summary of the main hormones detected in bovine milk Hormone
Ranges reported in bovine milka
Gonadal hormones Estrogens Progesterone Androgens
5–10 pg mL–1 2–20 ng mL–1 0–50 pg mL–1
Adrenal gland hormones Glucocorticoids (cortisosterone, cortisol) 5a-androstene-3,17-dione
0–50 ng mL–1 3 ng mL–1
Pituitary hormones Prolactin Growth hormone (GH)
5–200 ng mL–1 0–1 ng mL–1
Hypothalamic hormones Gonadotropin-releasing hormone (GRH) Luteinizing hormone-releasing hormone (LHRH) Thyrotropin-releasing hormone (TRH) Somatostatin Other hormones Parathyroid hormone-related protein (PH-rP) Insulin Calcitonin Bombesin (gastrin-releasing peptide) Erythropoietin Melatonin a
0.5–3.0 ng mL–1 0.5–3.0 ng mL–1 0–0.5 ng mL–1 10–30 ng mL–1 40–100 ng mL–1 5–40 ng mL–1 700 ng mL–1 0.25–450 ng mL–1 n/ab 5–25 pg mL 1
See text for details. Occurrence in milk suspected but no analytical data available.
b
by the mammary gland and excreted to milk. Their content in the mammary gland and in milk often exceeds that in the maternal blood plasma. This indicates that hormones in maternal milk may be involved, in situ, in the regulation of specific functions of the mammary gland, and they may also contribute to the growth of the newborn and to the development and maturation of its gastrointestinal tract and immune systems. Hormones found in milk could also regulate, at least temporarily, the activity of some endocrine glands until the newborn’s hormonal system reaches maturity (Bernt & Walker, 1999). Hormones already reported in milk are summarized in Table 1. The main categories to which these molecules belong are gonadal, adrenal, pituitary and hypothalamic hormones. Other molecules, such as proteins related to the parathyroid hormone, insulin, somatostatin, calcitonin, bombesin, erythropoietin and melatonin, also have been reported, and are discussed in this paper. The various ranges of concentrations for these hormones in bovine milk are included in the Table 1 for comparison purposes, but will be discussed throughout the present paper.
2. Gonadal hormones 2.1. Estrogens A number of techniques have been used to quantify estrogen content in milk, including colorimetry, spectrofluorometry, gas chromatography and high-pressure chromatography. However, the most reliable data were obtained using radioimmunoassay (RIA). This was used by Wolford and Argoudelis (1979) to estimate 17bestradiol (E2), estrone (E1) and estriol (E3) concentrations in milk and in a number of dairy products. Concentrations of E2 were found to be 10.3–14.4 pg mL–1 in raw milk and 5.071.2–9.079 pg mL–1 in skimmed milk. Concentrations of E1 were found to be 5.5–8.0 pg mL–1 in raw milk and 9.171.2–20.271.7 pg mL–1 in skimmed milk. These data show that approximately 65% of E2 and 80% of E1 can be found in the milk fat fraction. The occurrence of estrogens in both butter and skim milk clearly indicates a repartition of those steroids between the lipid and serum phases of milk. E1 is the predominant estrogen in milk. Its content is markedly higher than that of E2. In whey, 48% of E2 and 53% of E1 are bound to proteins. It is likely that these hormones are associated with bovine serum albumin since, in blood flow, these estrogens are transported by plasmatic serum albumin.
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Estrogens concentrations were also estimated using RIA by Monk, Erb, and Mollet (1975) during the gestation and the cycle. Before estrus, the average E2 content was 25.876 pg mL–1, increasing to 84741 pg mL–1 at estrus and decreasing to 42.4711.2 pg mL–1 after estrus. Average contents of E1 were 34.6679.8, 5876 and 5175.6 pg mL–1 during proestrus, estrus and metestrus, respectively. Concentrations of E2 and E1 found in milk were higher than those found in blood plasma throughout the ovarian cycle. They typically average 37.676 pg mL–1 for E2 and 4372 pg mL–1 for E1 in milk, whereas they average 1672 pg mL–1 for E2 and 1071 pg mL–1 for E1 in blood. Monk et al. (1975) also determined E2 and E1 contents in milk at three stages of gestation. The authors did not report any increase in estrogens during the cycle, but noticed an increase in plasmatic contents of E2 but not E1, contrary to what has been reported during the ovarian cycle. Estrogen concentrations are generally higher in milk than in the blood flow. This observation suggests the occurrence of an uptake of estrogens by the mammary gland. It also suggests that, since E1 content is higher than E2, the mammary gland either preferentially concentrates estrone or it might be able to convert, in situ, part of estradiol into estrone. Erb, Chew, and Keller (1977) have used RIA to determine the estrogen content in plasma and milk during the pre-partum period and lactation. During lactation (days 3–25), E2 concentrations were 2172 pg mL–1 in plasma and 13 pg mL–1 in milk. Contents of E1 were 5872 pg mL–1 in plasma and 2873 pg mL–1 in milk. These authors detected the 17a isomer (17a-estradiol), at a higher concentration in milk than in plasma (160714 and 15 pg mL–1, respectively). During the pre-partum period (days 3 to 1), E1, E2 and the epimeric form E2a represented 66%, 19% and 15% of total estrogens, respectively. Therefore, E1 is the predominant estrogen in milk. A similar pattern was found for plasma with E1, E2 and E2a representing 57%, 19% and 24% of total estrogens, respectively. During lactation (days 0–2), proportions of total estrogens represented by E1, E2 and E2a were, respectively, 55%, 19% and 26% in milk and 53%, 22% and 25% in plasma. During days 3–25 of lactation, E2 represented 7%, E2a 79% and E1 14% of total estrogens in milk, compared with 22%, 16% and 62%, respectively, in plasma. These data show that E2 contents are stable during the pre-partum period and lactation. On the contrary, E2a concentrations increased during lactation, whereas that of E1, which were high during the pre-partum period and first days of lactation (days 0–2), decreased thereafter (days 3–25 of lactation).
Harkness, 1974). This hormone was absent from colostrum. Its presence has been reported in milk approximately 15 days after parturition. Between days 15 and 57 of lactation, Pg concentrations in milk varied in a cyclic manner between 1.2 and 5.9 ng mL–1. Pg concentrations were higher in milk collected in the evening than in that collected in the morning. In addition to Pg, these authors have also reported the occurrence of 5a-pregnane-3,20dione, a metabolite of Pg, in many blood samples from pregnant cows. Milk Pg determination is now used for pregnancy diagnosis in cows (Comin et al., 2005). Pg is found in milk at concentrations of approximately 0.3–0.4 pg mL–1. Darling et al. (1974) also found that Pg concentrations are higher in cream than in skim milk. As for estrogens, Pg concentrations were found to be higher in milk than in blood plasma (Heap, Gwyn, Laing, & Walters, 1973). Ginther, Nuti, Wentworth, and Tyler (1974) have used RIA to determine Pg concentrations in cow milk during pregnancy on days 30, 60, 90, 120, 150, 180 and 210. It varied between 15.1 and 26.2 ng mL–1. These concentrations were generally four times higher than those in blood plasma (on average 21.2 ng mL–1 in milk compared with 5.3 ng mL–1 in plasma). Pg content in milk exceeds that of plasma already 4–5 days before and after calving (Erb et al., 1977). After calving, Pg content of milk decreases rapidly but remains higher than that of plasma and follows plasmatic variations of Pg. Pg contents have been determined in a number of dairy products (Ginther, Nuti, Garcia, Wentworth, & Tyler, 1976; Hoffmann, Hamburger, & Karg, 1975). Both studies reported similar data. According to Ginther et al. (1976), whole milk, skim milk and cream contain 11.376, 4.674 and 58.775.3 ng mL–1 of Pg, respectively. As for the commercial products, concentrations were 9.575 ng mL–1 in whole milk, 2.176 ng mg–1 in skim milk, 72.775.8 ng mL–1 in cream and 132.975.1 ng mL–1 in butter. The values were 98 ng mL–1 in whole milk powder and 17 ng g–1 in skim milk powder (Hoffmann et al., 1975). The only discrepancy reported concerned butter for which a value of 132.975.1 ng g–1 in Pg was reported by Ginther et al. (1976) and 300 ng g–1 by Hoffmann et al. (1975).
2.2. Progesterone
3. Adrenal gland hormones
Progesterone (Pg) concentrations have been determined first in bovine plasma by gas chromatography during pregnancy and after parturition (Darling, Laing, &
The occurrence of corticosteroids in cows’ milk has been demonstrated for the first time in the late 1950s (Ratsimamanga et al., 1956). According to Gwazdauskas, Pappe, &
2.3. Androgens Very few studies have been published on androgen content of milk. Testosterone in milk has been determined by RIA after ether extraction and purification on a silica gel. The data reported varied from non-detectable to 50 pg mL–1 in milk at estrus and 150 pg mL–1 during the luteal phase. The ratio between free and conjugated testosterone is 1/1 (Hoffman & Rattenberger, 1977).
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Mc Gilliard, (1977), glucocorticoid concentrations in milk vary between 0.7 and 1.4 ng mL–1. There is no difference between whole and skim milk. Corticosteroı¨ d contents of milk are much lower than those of blood plasma. According to Tucker and Schwalm (1977) milk cortisol represents only 10–23% and corticosterone 60–90% of their plasmatic homologs. Injections of ACTH to cows cause a temporary increase in milk glucocorticoids. Similarly, administration of hydrocortisone generates an increase of glucocorticoids in milk (Gwazdauskas et al., 1977). Cortisol and corticosterone are the main glucocorticoids in blood plasma of cows. Their detection has been achieved by a radio-binding assay after extraction from the milk using ethyl acetate and chromatographic separation using Sephadex LH-20 (Tucker & Schwalm, 1977). During lactation (0–11 months), glucocorticoid concentrations in milk only represent 4% of the blood plasma concentrations, indicating that only a small amount is transferred to the milk. However, the relative proportions of the different glucocorticoid hormones differ between blood plasma and milkrent. For instance, cortisol is predominant in blood plasma (66% of total glucocorticoids) whereas corticosterone represents 90% of total glucocorticoids in milk. During lactation, glucocorticoid concentrations in milk decrease gradually and significantly. They go from 0.5970.11 ng mL–1 during the first 2 months, to 0.287 0.04 ng mL–1 between 5–7 months and 0.2570.02 ng mL–1 between 9 and 11 months of lactation. This decrease is essentially related to changes in cortisol concentrations, while corticosterone concentrations slightly increase during the same period. Contrary to estrogens, glucocorticoids are not concentrated in cream. Indeed, 71–89% of the milk estrogens will be found In the cream fraction, whereas it contains only 12–15% of the corticosteroids. In cows’ milk, corticosteroids are equally distributed between caseins and whey protein fractions. Gorewit and Tucker (1976, 1977), studying the binding of glucocorticoids in the mammary gland of lactating cows, have demonstrated the presence of glucocorticoid receptors. Sections of mammary glands were incubated for 1 h, at 37 1C, in the presence of cortisol-3H or hexamethasone-3H. The repartition of the compound between supernatant and pellet of a 700 g centrifugate showed the high affinity of glucocorticoid receptors in the mammary gland of the lactating cow. It was estimated that the binding constant (Kd) with cortisol was between 10–8 and 10–10 M. Also, it has been estimated that each mammary cell contained approximately 7500 high-affinity binding sites (Tucker & Schwalm, 1977). Glucocorticoids possibly act in conjunction with other hormones to maintain lactation. Their effects would be mediated by specific receptors. Glucocorticoids also seem to reduce, in a dose-dependent manner, glucose uptake by the mammary gland. Glucose is an important substrate for lactose production. Lactose content may therefore be
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regulated by glucocorticoids, which explains the decrease of milk production when glucocorticoids content is high. The steroid 5a-androstane-3,17-dione originates from the reduction of androstenedione following the action of 5a-oxydo-reductase, in the surrenal glands. Darling et al. (1974) achieved its isolation in milk by gas chromatography. However, it was only found in 3 out of 12 colostrum samples. This steroid appears in milk right after calving. Between days 4 and 57 of lactation, the average content of androstanedione in milk is 3 ng mL–1 whereas during pregnancy (3–136 days), it is 270.09 ng mL–1 of milk. This concentration remains constant throughout pregnancy. Higher concentrations of this hormone are found during the evening compared to the morning milking. 4. Pituitary hormones 4.1. Prolactin Prolactin has been detected first in cows’ milk using RIA with a double antibody (Malven & Mc Murtry, 1974), with contents varying from 5 to 200 ng mL–1. However, the numerous milk samples analyzed by the authors allowed the determination of an average value at 50 ng mL–1. Gala, Forsyth, and Turvey (1980) have determined the prolactin content of five milk samples using RIA after purification on a Sephadex G100 column. This column pre-purification allowed the recovery of most of the prolactin in a homogeneous peak. Prolactin content varied between 3.7 and 14.2 ng mL–1. The bioactivity of milk prolactin has been demonstrated in vitro on explants of mammary glands from pseudopregnant rabbits (Gala et al., 1980). Prolactin concentrations appear to fluctuate seasonally and are higher in summer than in winter. Storage of milk at 4 1C does not affect prolactin concentrations whereas 59% is lost upon storage at 15 1C (Malven & Mc Murtry, 1974). Part of the prolactin content of milk is associated with milk fat globules since a centrifugal separation of fat removes also 60% of the initial prolactin content of milk (Malven & Mc Murtry, 1974). The prolactin content of colostrum is much higher than that of milk, varying between 500 and 800 ng mL–1, compared to 6–8 ng mL–1 in milk (Kacsoh et al., 1991). Prolactin in milk probably originates from blood plasma. Its biological functions are not clearly established. It could stimulate lactation by a direct action on secretory cells. In the newborn, the permeability of the gastro-intestinal tract allows the passage of prolactin from the intestine to the blood. In the adult, prolactin is probably hydrolyzed in the intestine. 4.2. Growth hormone The growth hormone (GH) or somatotropin, in milk was first detected using RIA (Torkelson, 1987) at concentrations lower than 1 ng mL–1. Treatment of early lactating
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cows with bovine somatotropin (bST) does not induce significant increases in the milk concentration of this hormone (de Boer, Robinson, & Kennelly, 1991). GH is thought to be acting on the mammary gland by means of specific receptors as shown by Glimm, Baracos, and Kennelly (1988). It was also found that GH increased the concentration of insulin-like growth factor-1 (IGF-I) in epithelial cells of the mammary gland of lactating cows (Glimm et al., 1988). 5. Hypothalamic hormones 5.1. Gonadotropin-releasing hormones Gonadotropin-releasing hormone (GnRH) was detected and quantified in cows’ milk by Baram, Koch, Hazum, and Frikin (1977). After acid precipitation of caseins from reconstituted skim milk powder, GnRH was extracted by an acid-methanol procedure and analyzed by RIA. Concentrations of GnRH in milk vary between 0.5 and 3 ng mL–1. GnRH extracted from milk has similar biological activities as the hypothalamic hormone since it induces the release of LH and FSH from rat pituitary glands incubated in vitro. GnRH content in milk is 5–6 times greater than in blood plasma. GnRH might be from an extra-hypothalamic origin but it is more likely coming from an active transport by the mammary gland. In the newborn, GnRH is absorbed by the intestine in an active form. It may be involved in the masculinization of the male hypothalamus by stimulating androgens secretion that would act on the brain. Gonadotropin-releasing hormone-associated peptide (GAP) was also found in bovine colostrum, using RIA (Zhang et al., 1990). This 56-amino acids peptide has a sequence identical to the C-terminal end of GnRH. GAP could therefore be the precursor of GnRH. GAP concentration in defatted colostrum is 1.570.1 pmol g–1. GAP also induces the liberation of LH and FSH from rat pituitary glands incubated in vitro. According to Zhang et al. (1990), GAP could be synthesized in the mammary gland. 5.2. Luteinizing hormone-releasing hormone (LH-RH) LH-RH content in milk and colostrum has been determined by RIA, after methanol-acid extraction and HPLC (Amarant, Fridkin, & Koch, 1982). In the colostrum, LH-RH concentration is 11.7870.72 ng mL–1, whereas in milk it varies between 0.5 and 3 ng mL–1. Milk or colostrum contents of LH-RH exceed that of blood plasma. The origin of LH-RH in milk is still unknown. This hormone may originate from the blood and be concentrated in the mammary gland by an active process or be of an extra-hypothalamic origin. LH-RH from milk is biologically active. It induces the liberation of LH from rat pituitary glands incubated in vitro. LH-RH is absorbed in intact and active form by the newborn’s intestine. Milk
could be considered as a source of LH-RH for the newborn stimulating the secretion of the pituitary gonadotropins. 5.3. Thyrotropin-releasing hormone Thyrotropin-releasing hormone (TRH) has been measured in milk and colostrum by RIA (Amarant et al., 1982), using a procedure similar to that of LH-RH. TRH concentrations in colostrum and milk are 0.1670.03 and 0.05 ng mL–1, respectively. Both colostrum and milk show higher concentrations of TRH than blood plasma. As for the other hypothalamic hormones, the origin of TRH in milk is unclear. TRH is absorbed in intact and active form by the newborn’s intestine. The intestinal hydrolysis of TRH is low during the first 3 weeks after birth. It is possible that TRH modulates TSH secretion in the newborn. 5.4. Somatostatin The occurrence of somatostatin in cows’ milk has been demonstrated by enzyme immunoassay (EIA) on defatted, decaseinated milk (Takeyama et al.,1990). Somatostatin concentrations in milk vary between 10 and 30 pmol L–1. It does not seem to be affected by parturition. 6. Other hormones 6.1. Parathyroid hormone-related protein Many authors have mentioned that parathyroid hormone-related protein (PTH-rP) is present in cows’ milk. The RIA analysis of this hormone was possible after partial purification by affinity chromatography using Sepharose gel coupled with polyclonal antibody anti-PTH 1-34. The PTH-rP content in milk has been estimated to 95.6734 ng mL–1 (Budayr et al., 1989). Ratcliffe et al. (1990) obtained similar results on both fresh and pasteurized milk (86.8720.8 ng mL 1 and 73.377.1 ng mL–1), respectively. This suggests that PTH-rP is, to some extent, heat-stable and not affected by pasteurization. Purified extracts of PTH-rP were analyzed by SDS-PAGE and immuno-blotting. Two biologically active molecular forms (27 and 21 kDa) were detected (Ratcliffe et al., 1990). Law et al. (1991) reported PTH-rP concentrations in whole and pasteurized milk as 59.2718.5 and 66.77 12.5 ng mL–1, respectively. The breed of cow seems to have an influence on PTH-rP content, since milk from Jersey cows contained 52.675.4 ng mL–1 whereas that from Friesians cows had 41.074.8 ng mL–1 (Law et al., 1991), and this difference was statistically significant (Po0,01). PTH-rP content of milk increased gradually during lactation (Goff, Reinhardt, Lee, & Hollis, 1991). PTH-rP contents do not seem to vary in commercial dairy products. Budayr et al. (1989) showed that the PTH-rP content was 81.4717.9 ng mL–1 in whole milk, 88.978.7 ng mL–1 in low-fat milk, 118.3718.9 ng mL–1 in
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skim milk and 76.2732.7 ng mL–1 in reconstituted skim milk powder. The physiological functions of PTH-rP have not been clearly established. Produced by the mammary gland, this hormone might be involved in the transport of calcium from blood plasma to milk. It would therefore ensure the calcium supply to the newborn. However, this hypothesis is disputable since Goff et al. (1991) found no correlation between PTH-rP concentrations and calcium content in milk. PTH-rP also may be involved in the maturation of intestinal epithelium in intestinal motility. 6.2. Insulin Insulin content in colostrum is between 0.67 and 5.0 nM, which is 100-fold higher than the concentration in the blood plasma (Ballard, Nield, Francis, Dahlenburg, & Wallace, 1982). According to Malven (1977), insulin concentrations in milk varied from 37.1714 ng mL–1 during the pre-partum period, to 6.272.1 ng mL–1 during post-partum and 5.570.6 ng mL–1 after parturition.
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concentration (5–25 pg mL 1). Its concentration in milk shows a diurnal pattern (maximal concentration at midnight and minimal concentration at noon), which parallels that of serum to some extent. It has been suggested that night-time milk could serve as source of melatonin to improve sleep and diurnal activity in elderly people (Valtonen, Niskanen, Kangas, & Koskinen, 2005). 7. Conclusion Bovine milk and milk products contain a number of hormonal substances originating from the cow’s blood flow or synthesized in the mammary gland. Although occurrence of these hormones in milk may benefit the newborn, it is not clear whether they can bring health benefits to humans in milk products. There is a need to update the data concerning hormonal levels in milk and milk products, especially in the light of changes in the genetic background of dairy cattle in the last decades, as well as in animal feeding and husbandry and new processes that have emerged in milk industry.
6.3. Calcitonin Calcitonin concentrations in human milk has been estimated at 700 ng mL–1 using RIA (Koldovsky, 1989). Calcitonin inhibits the liberation of prolactin. 6.4. Bombesin Bombesin (gastrin-releasing peptide) is a 14 amino acids peptide that is known to influence the gastric hormonal secretions following ingestion (Lazarus, Gaunido, Wilson, & Erspamer, 1986). Satiety, blood sugar concentrations, gut acidity and concentrations of some gastro-intestinal hormones are known to be influenced by bombesin. Bombesin has been found in human milk, cows’ milk, milk powder and whey (Lazarus et al., 1986). Concentrations in human, bovine and porcine milks range from 0.25 to 450 ng mL–1 (Koldovsky, 1989). 6.5. Erythropoietin Human milk is known to contain erythropoietin (Grosvenor et al., 1993). It has been suggested that erythropoietin is being transferred from the mother to the offspring by the milk and that it might be able to stimulate erythropoiesis in the offspring (Grosvenor et al., 1993). No analytical data are available for cows’ milk. 6.6. Melatonin Melatonin is a hormone synthesized by the pineal gland in a diurnal pattern reflecting photoperiodicity. Melatonin has been found in human, bovine and goat milk (Eriksson, Valtonen, Laitinen, Paananen, & Kaikkonen, 1998; Valtonen, Kangas, Voutilainen, & Eriksson, 2003) at a low
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