Download Possible Physiological Role of Hormones and Hormone

Biology of Human Milk, edited by Lars A.
Hanson. Nestle Nutrition Workshop Series,
Vol. 15. Nestec Ltd., Vevey/Raven Press,
Ltd., New York © 1988.
Possible Physiological Role of Hormones
and Hormone-Related Substances Present
in Milk
O. Koldovsky, A. Bedrick, P. Pollack, R. K. Rao, and
W. Thornburg
Perinatal and Nutritional Sciences Section, Departments of Pediatrics and
Physiology and Children's Research Center, University of Arizona, Health
Sciences Center, Tucson, Arizona 85724
The presence of hormones in milk was described 50 years ago (1-3), and
important studies were performed in the late 1950s (4,5); but only in the past
decade have methodological advances in hormone assays allowed detailed
exploration of this subject. In this presentation we deal with two main questions: (a) Which hormones are present in milk?, and (b) Are some absorbed
in active form from the gastrointestinal tract of suckling animals?
HORMONES DETECTED IN MILK
Hormones detected in milk are listed in Table 1. Concentrations in milk
versus concentrations in plasma vary substantially for different hormones.
Table 2 lists the hormones with a significantly higher concentration in milk
than in blood. Some hormones, such as (T3) (tri-iodothyronine), progesterone, and prolactin, have ratios between 0.5 and 1.0; (T4) (thyroxine) and
estradiol, as well as binding proteins for progesterone, corticoids, and thyroid hormones, are found in milk at very low concentrations (between 1%
and 10% of serum). It is important to note that during lactation, many hormones decrease in concentration in milk.
Many questions are posed by the data summarized in Tables 1 and 2, but
our comments are concentrated mainly on thyroid hormones, nucleotides,
and gastrointestinal hormones and on questions relating to the presence of
insulin, epidermal growth factor, and prostaglandins.
Thyroid Hormones
Considerable attention has been given to the presence of thyroid hormones
in milk (6-15). Some authors (9,14,16) ascribe only a minor role to milk
123
124
PHYSIOLOGICAL ROLE OF HORMONES
TABLE 1. Hormone and hormone-related substances in milk or colostrum*
Substance"
Human
Cow
Rat
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Prolactin
GnrH
Ovarian steroids
Adrenal steroids
TRH
TSH
T4
T3
Calcitonin
EGF
Erythropoietin
Insulin
Cyclic AMP
Cyclic GMP
PG
Relaxin
Neurctensin
Somatostatin
Bombesin
X
X
X
X
X
X
X
Horse, sheep, goat, pig
Horse, dog (?)
Horse, sheep
Goat
X
Mouse
Mouse, sheep
X
Goat, guinea pig
X
X
X
Other mammals
X
X
X
Sheep
X
" Adapted from refs. 94 and 95.
* (GnrH) gonadotropin-releasing hormone; (TRH) thyroid-releasing hormone; (TSH) thyroid-stimulating hormone.
TABLE 2. Milk/plasma concentration ratio
Substance
Species
Ratio
Prolactin
GnrH
Calcitonin
Cow"
Human
Human
Human
Mouse
Human"
Rat, human
Human
5-10
96
5-6
97-99
EGF
Insulin
Cyclic AMP, cyclic GMP
PG
Cow
Relaxin
Neurotensin
Somatostatin
" Colostrum.
* Absent in infant formulas.
Human
Human
Human
Sheep
20-90
100-5,000
50-200
100
8-20
100
0.5-10
8-higher
5-10
5
3
Ref.
100
77," 78, 81, 87," 101, 102
85, 103-105
106
34
37, 38," 43, 107
40, 41, 43
108
109
110
110
PHYSIOLOGICAL ROLE OF HORMONES
125
thyroid hormones because of the low amounts detected in milk, although
the reported values for concentrations of T3 and T4 in human milk vary
substantially among different laboratories, probably due to procedures of
milk extraction (17). Others report that breast-feeding and the consequent
availability of thyroid hormone in the milk for gastrointestinal absorption
prevented sequelae of neonatal hypothyroidism, both experimentally in monkeys (8) and in hypothyroid children (6,13). Cow's milk-based formulas contain only very low quantities of thyroid hormone (6,11). Some authors consider milk a source of iodine for thyroid function (18-20).
Nucleotides
Nucleotides, some directly related to hormonal activity, have attracted
the attention of many researchers. Several papers have reported the presence
of acid-soluble nucleotides in milk and colostrum: human (21-25); bovine
(22,24,26-31); goat (24,26-28,30); and sow (26,32). Thermal industrial processing decreases the concentration of acid-soluble nucleotides in milk (31).
Comparison of the data indicates a few similarities but many differences in
representation of individual nucleotides in milk and colostrum of various
species.
Cyclic AMP has been found in human (22), bovine (22,28), goat (28), and
rat milk (33,34), and in colostrum and milk of guinea pig (35). Cyclic GMP
is present also in human milk (34). The fate of cyclic AMP and cyclic GMP
in milk after oral intake is not known. Skala et al. (34) have shown that
concentration of cyclic AMP in stomach content of suckling rats was similar
to that in fresh rat milk. Preliminary experiments indicate the possibility that
cyclic AMP orally administered to suckling rats is absorbed into the circulation (34).
Because of differences in composition and quantity of nucleotides in milk
and infant nutrition formulas, Gil and Molina (36) performed studies comparing the effect of feeding healthy newborns with human milk, infant milk
formula, and nucleotide-supplemented milk formula during the first month
of life. Based on the determinations of lipoprotein patterns as well as the
changes of the plasma fatty acid patterns, they concluded that nucleotides
of human milk may be involved in lipoprotein synthesis during the neonatal
period.
Gastrointestinal Hormones
Table 1 lists the presence of several gastrointestinal hormones. Little is
known about their physiological role.
126
PHYSIOLOGICAL ROLE OF HORMONES
STUDIES ON THE GASTROINTESTINAL PROCESSING OF
HORMONES AND HORMONE-RELATED SUBSTANCES PRESENT
IN MILK
Prostaglandins
The presence of prostaglandins (PG) has been demonstrated in human
(37-39), bovine (40-41), and rat milk (42). Reid et al. (38) did not find PG
in infant formulas, but according to Materia et al. (43), they are present in
commercially available bovine milk. Two studies have explored the stability
of PG in milk. In vitro studies by Mann (40) have shown that incubation of
bovine milk at 37°C for 6 hr does not cause a loss of endogenous or added
PG. The higher stability of PG in breast milk is confirmed by the low ratio
of milk PGF metabolites to PGF (0.3-0.5) versus that in plasma (approximately 15-16). This raises the interesting possibility that PG survival in milk
is great enough to exert an effect on the neonate (37,38,43).
In our first study (44), we demonstrated that after gastric administration
of radiolabeled PGE2 to suckling rats, authentic PG could be found 30 and
60 min later in intestinal lumen and wall, as well as in liver and kidney
homogenates. In other studies, PGF2 was fed gastrically (45) to suckling and
weanling rats. The mass of radioactive PGF2 administered to sucklings was
similar to the average daily consumption of PG in suckling rats (44). Two
hours after administration, livers were analyzed for total radioactivity recovered by organic solvent extraction. Recovery of total counts in the liver
of sucklings was considerably higher in sucklings than in weanlings [sucklings: N = 10, 11.0% ± 1 . 1 (SEM); weanlings: N = 7, 3.3% ± 0.5 (SEM)].
Organic solvent extracts were then subjected to thin-layer chromatography.
After exposure to iodine vapor, distribution of tissue radioactivity (intact
prostaglandin and metabolites) was determined by scraping each spotting
channel into fifteen 1-cm fractions from the spotting origin to solvent front.
Scrapings were transferred to plastic counting vials, and scintillation counting was performed.
Figure 1 depicts a typical thin-layer chromatography distribution pattern
of radioactivity (intact compound plus metabolites) present in the liver of
suckling and weanling rats. Three distinct areas of radioactivity appeared.
The first, identified as area I and migrating in the third fraction, was defined
as radioactivity associated with authentic PGF2 standard (unmetabolized
prostaglandin). Area II migrated in scraping fractions 4 to 6, and area III in
fractions 8 to 10.
Calculation of average values from all animals studied showed no significant age differences in area III; however, a greater percentage of the label
was present as authentic PGF2 in the livers of sucklings (11.0% ± 1.1) compared to weanlings (7.0% ± 1.1). Of the original dose given to each animal,
there was a sixfold difference in the presence of unmetabolized PGF 2 in
PHYSIOLOGICAL ROLE OF HORMONES
127
WEANLING
T
0
FIG. 1. Typical thin-layer chromatographic distribution of radioactive PG and its metabolite
extracts of liver homogenates of
suckling and weanling rats 2 hr
after oral PG administration.
(From ref. 45.)
A
L
0
U
N
S
ORIGIN
AREttS
3
5 7 9 11 13 15
FRONT
P G F ^
"-^
I
II
III
SCRAPING FRACTIONS
sucklings versus weanlings (1.2% ± 0.2 vs. 0.2 ± 0.1). There was also a
significantly higher percentage of radioactivity present in area II in sucklings
(43.3% ± 1.6) than in weanlings (34.3% ± 3.0).
The developmental changes in intestinal metabolism of PGF2 were examined in vitro, utilizing the everted sac technique (Fig. 2). Mucosal fluid
(outside) initially contained [3H]PGF2. Suckling animals had a greater capacity for transfer of radioactivity into serosal (inside) fluid. Qualitative characterization of PG and its metabolites, as described above, showed that
compared to the weanlings, suckling animals had a greater proportion of
radioactivity present as intact unmetabolized PGF 2 present in tissue of all
segments; however, weanling animals had more PGF2 degradation products
of lesser polarity than suckling animals.
The studies discussed above suggest that suckling and weanling rats differ
considerably in processing orally administered PG. Other studies have
shown that the inhibition of intestinal PG synthesis by indomethacin does
48
SU
38
NE
1
1- 26
18
t
1
h i
I
II III IU U
1-2 PGF2A 4-6 PGH8-11
EVERTED SACS
FIG. 2. Distribution of radioactivity from a chromatogram of middle intestinal segment (everted
sacs) of suckling (SU) and weanling (WE) rats. Incubation time: 60 min. Ordinate: Area I is defined
as the first and second fraction of the chromatogram; this region is more polar than PGF2». Area II
is the third fraction (authentic PGF2ct). Area III consists of fractions 4-6, regions between PGF2o,, and
13,14-dihydro-15-keto PGF2(1 (PGM). Area IV is fraction 7, the migration region of PGM. Area V consists
of fractions 8-10, chromatographic regions less
polar than PGM. Abscissa: The percent of total radioactive counts present in the tissue segment in a
particular chromatographic region. Bars and short
vertical lines denote mean and SEM values, respectively; /V/group = 6; (*) statistical significance
between corresponding values of suckling and
weanlings. (From ref. 111.)
128
PHYSIOLOGICAL ROLE OF HORMONES
not result in gastrointestinal ulceration in suckling rats or in rats prevented
from weaning. This is in contrast to the jejunoileal ulcerations found in indomethacin-treated weaned and adult rats (42). The presence of PG in milk
may exert a cytoprotective effect, but the specific mechanisms involved are
unclear. PG in milk has been shown to be effective in promoting healing of
peptic ulcers and in protecting the gastroduodenal mucosa against experimentally induced ulcers (43). In addition, administration of oral PGE2 to
humans has been demonstrated to protect the gastrointestinal mucosa from
blood loss induced by indomethacin (46); to inhibit gastric acid secretion in
humans (47); and to exhibit trophic action on the rat intestinal mucosa (48).
In addition to PG, other substances present in breast milk may be cytoprotective for the gastrointestinal epithelium. Lichtenberger et al. (49,50) have
concluded that PG-induced cytoprotection was mediated in part by localized
increases in phospholipid concentration.
Protein or Peptide Hormones
Extensive literature exists demonstrating that the gastrointestinal tract of
suckling mammals possesses the ability to absorb various proteins with substantial preservation of their immunological properties (51-55). In agreement
with these studies, the absorption of large molecular hormones from the
gastrointestinal tract has been demonstrated in suckling rats and mice. This
was evaluated by following the changes in concentration in serum and/or
other tissues of melatonin (56) or prolactin (57,58), or by determining physiological changes in experimental animals caused by erythropoietin (59-61),
thyroid-stimulating hormone (TSH) (62), or (ACTH) (63). We shall discuss
in detail data on insulin and epidermal growth factor (EGF).
Insulin
The presence of insulin in human colostrum and milk has been demonstrated by several laboratories (64-66). The first experiments to test its oral
hypoglycemic effect in suckling rats were performed in our laboratory in
Prague about 30 years ago. In suckling rats, a considerable decrease in blood
glucose levels was observed; no effect was seen in weaned rats (67). Since
the administration of insulin directly into the jejunum or ileum of both suckling and adult rats caused a similar decrease in blood glucose levels (68),
we concluded that the absence of absorption of insulin in a biologically active
form in weanling rats was due to the development of gastric digestion. This
conclusion is further supported by our original observation of low proteolytic
activity in stomach homogenates of suckling rats, compared to the high
activity in 21- and 30-day-old rats (67). The hypoglycemic effect of orally
PHYSIOLOGICAL ROLE OF HORMONES
129
administered insulin has also been reported in other species, such as piglets
(69) and calves (70).
Epidermal Growth Factor
Epidermal growth factor (EGF) was discovered by Cohen in the 1960s
(71). It is produced in the salivary and Brunner's gland, and is mitogenic in
vivo and in vitro (72). It is noteworthy that EGF is trypsin, chymotrypsin,
and pepsin resistant. The presence of EGF and possibly other growth factors
in colostrum and milk of various species was indicated by the growth-promoting effect of colostrum on the gastrointestinal tract of newborn pigs (73).
A similar effect was also seen in dogs (74) and rats (75). Other studies demonstrated the presence of EGF and related substances in colostrum and milk
of various species using radioimmunoassay (RIA), radioreceptor assay
(RRA), or determination of mitogenetic activity using various cell lines in
vitro [human (66,76-82); bovine and goat (83); rat (84); and mouse (85)].
Before discussing some of the results obtained in our laboratory, there
are a few points to make concerning the EGF levels in colostrum and milk.
First, it is important to realize that the results obtained in the three methods
of determination mentioned above are quite dissimilar. RRA-positive material can represent a mixture of RIA-positive material and other non-EGF
substances. Similarly, mitogenic-positive material can also be a mixture of
various growth factors. Second, values in both colostrum and milk exceed
values found in corresponding plasma, but the postnatal changes in the concentration of EGF in milk are not the same in all species. In humans, the
EGF concentration (measured by specific RIA) decreases considerably postnatally (81). In mouse, however, using specific RIA, there is an increase in
EGF serum concentration in the middle of the suckling period, the values
at birth and at the end of the third week of lactation being approximately 5
to 6 times lower (85). Third, it is perhaps of potential clinical significance
that several infant formulas do not contain EGF (SMA, Similac, Nutramigen,
Isomil, Isocal) (77,86).
In our laboratory, we first studied the gastrointestinal processing of 125Ilabeled mouse EGF given via gastric tube to suckling rats (84). We used
Sephadex G-25 column chromatography, which separates EGF and its metabolites into three peaks: peak A, authentic EGF (or EGF with minor
changes) present in the void volume; peak B, a mixture of low-molecularweight peptides and free iodine present in the column volume; and peak C,
a mixture of mono- and diiodotyrosine. Experiments have shown that acid
extracts of contents of the stomach and intestinal lumen contain "intact"
EGF and very few degradation products within 30 or 60 min after EGF
administration. In the stomach and intestinal wall, EGF is processed in a
time-dependent fashion; the stomach produces peak B, and the small intestine peak C (Figs. 3 and 4).
130
PHYSIOLOGICAL ROLE OF HORMONES
Stomach
Wall
i
o
FIG. 3. Distribution of radioactivity in the stomach and intestinal wall 30 min after gastric
administration of 125I-EGF to
suckling rats. (From ref. 84.)
OOOOOOQOO
10
20
30
40
Fraction Number
Stomach
Wall
FIG. 4. Distribution of radioactivity in the stomach and intestinal wall 60 min 12S
after gastric
administration of
I-EGF to
suckling rats. (From ref. 84.)
•oonnPOooooooooo
10
20
Fraction Number
30
40
PHYSIOLOGICAL ROLE OF HORMONES
131
WALL
*
30m i n
A IR RE:
SUCKL WEANL.
* = S I G H . ..... •=.. C 0 R R E S P 0 N D I N G S U C K L I H G
WALL
IR RE
B
*=S I GH . A G . C0RRESP0N D I N G SUCKL I N G
*=S I GN . AG . ST0MA C H U ALUE S
H=6 ; 3O M I N
FIG. 5. Characterization of 125I-EGF in the stomach content and wall (A) and in the small
intestinal content and wall (B) of suckling and weanling rats 30 min after gastric administration of 12SI-EGF. N/group = 6 except when the minus sign is above the column, then
only two values are presently available. In this case, vertical lines denote the higher value.
(A) peak A; (IR) immunoreactive material; (RB) receptor bound; ($) value significantly
different from corresponding value of the stomach; (*) significant difference from corresponding values of suckling; (") significant difference from the value of peak A. (From
Thomburg et al., unpublished data.)
Further studies were performed to compare suckling and weanling (29
days old) rats (87); animals were killed 30 min after administration of EGF.
Figure 5 depicts the effect of weaning on gastric processing of EGF. Three
criteria were used: (a) the amount of EGF present in peak A on Sephadex
G-25 column chromatography (A); (b) the amount of immunoreactive (IR)
EGF; and (c) the amount of EGF binding to A431 cell receptors (RB). All
132
PHYSIOLOGICAL ROLE OF HORMONES
values were expressed as percentages of the original EGF administered. In
stomach wall and content, weaning animals had decreased values of these
measures, and the decrease found in the small intestinal contents and wall
was even more pronounced (Fig. 5). The data also show a quantitatively
different decrease in each of the three measures. We found that despite the
changes in gastrointestinal processing of EGF with weaning, intact EGF was
still delivered to peripheral tissues [kidney, liver (data not shown)].
Another group of experiments was performed to explore the locus of EGF
absorption (88). 125I-EGF was injected into ligated stomach, jejunal, or ileal
segments of intestine of anesthetized 12-day-old suckling or 30-day-old
weanling rats. The abdomen was sutured, and after 60 min at 33°C, the
animals were killed and the appropriate tissues and lumen analyzed for the
remaining unabsorbed 125I radioactivity. It was shown that the intestine is
primary site of gastrointestinal absorption of 125I-EGF; absorption from the
stomach is negligible.
Other experiments have characterized the handling of EGF by the intestine
during development (89). The in vitro method that we used (everted sacs)
enabled us to compare the role of two distinct intestinal segments. The transfer of EGF was studied in jejunum and ileum of suckling (14 days old),
weanling (30 days old), and adult (3 months old) rats. The mucosal (outside)
fluid initially contained 0.33 HIM 125I-mEGF. Mucosal fluid, intestinal wall,
and serosal fluid (inside) were analyzed for total radioactivity. Acid extracts
were subjected to immunoabsorbent and Sephadex G-25 column chromatography. Transfer of total radioactivity into serosal fluid decreased with
age (by 80%), but due to a relatively lower loss of EGF immunoreactivity
in the preparations from weanling and adult rats, transfer of total immunoreactive EGF to the serosal fluid decreased with age by only 30%. In the
weanling and adult rat, no differences between jejunum and ileum were
observed, whereas in the suckling rat the degradation and transfer were
higher in jejunum than in ileum. These in vitro results clearly demonstrate
that the small intestine possesses the capability to transfer EGF. It is noteworthy that this capability was also found in adult animals. Further studies
will be performed to characterize the mechanisms of transfer.
We have described various effects of EGF on the gastrointestinal tract
when given orally or parenterally in pharmacologic doses (for review, see
refs. 90 and 91). To test its oral effect in suckling rats in physiologic doses,
11- to 14-day-old rats were hand gavage fed an artificial milk diet with or
without added EGF every 3 hr (92). Intake was adjusted to deliver 300
kcal/kg body weight/day formula and 16 n-g/kg body weight/day EGF, approximating the daily energy inake and about twice the estimated daily EGF
intake for suckling rats (84). Weight gain during the experimental period did
not differ between groups (EGF 3.8 ± 0.2 g; control 3.7 ± 0.1 g). Proximal
and distal colons were analyzed for total protein and DNA content and
protein/DNA ratio.
TABLE 3. Effect of EGF fed in formula on growth of colon of 14-day-old rats*
Proximal segment
EGF
P
4.5 ± 0.2
D
242.9 ± 7.9
P/D 18.6 ± 0.9"
Distal segment
Total colon
Controls
EGF
Controls
EGF
Controls
5.5 ± 0.5
220.6 ± 14.0
25.7 ± 2.8
2.9 ± 0.02c
199.8 ± 10.6c
14.3 ± 0.4c
5.0 ± 0.02
129.5 ± 1 1 . 3
41.1 ± 2.4
7.4 ± 0.02c
442.7 ± 14.2
16.7 ± 0.5c
10.5 ± 0.5
354.7 ± 19.8
30.5 ± 2.1
• Means ± SEM; 13-16 rats/group; (P) mg total protein; (D) \i.g total DNA; (P/D) protein/DNA. (Data from
ref. 92.)
" p < 0.05.
c
p < 0.01 vs. control by unpaired Mest.
134
PHYSIOLOGICAL ROLE OF HORMONES
Data summarized in Table 3 show that when EGF was delivered enterally
in physiological quantities with an artificial formula, it increased the DNA
content and reduced the protein content of suckling rat colon. These data
support a physiological role for breast-milk EGF in gastrointestinal
development.
CONCLUSION
Many hormones and hormone-related substances are present in human
milk and in milk of various species. Their concentration varies with species
and duration of lactation. To approach the question of the physiological
significance of their presence in milk, we have summarized some of the data
obtained in our laboratory on gastrointestinal processing of PG, insulin, and
EGF in the developing rat. Further experiments are needed to analyze these
processes in more detail, with the inclusion of comparative studies of other
species to determine if these findings are applicable to humans. The question
posed several years ago (93), namely, whether we should consider hormones
in milk "equally" important components of infant nutrition as the major
nutrients, remains open for further investigation.
ACKNOWLEDGMENT
This work was supported by National Institutes of Health Grant AM27624
and Nestle Nutrition Research Grant Programme 84/04. We are grateful to
Miss Melita Stine for skillful help with references.
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DISCUSSION
Dr. Lucas: There are a large number of hormones in milk with multiple effects,
but does it not disturb you that when you remove all, or nearly all, these hormones
PHYSIOLOGICAL ROLE OF HORMONES
139
fron the infant's diet by formula-feeding, there is no obvious abnormality in the
adaptation to extrauterine nutrition, no obvious clinical biochemical abnormality,
andno abnormality in subsequent growth?
Z>. Koldovsky: It is a puzzle, I agree; however, we did our experiments between
days 10 and 14, after the early neonatal period, and maybe the supply of hormones
in olostrum is the important thing. Also, every organism has alternative pathways.
Since we may be able to compensate with another pathway, we may not see the
expected ill effects. Whether it is good or bad to activate these alternative pathways,
I dc not know.
D-. Guesry: Could you comment on the importance of the mother's diet with regard
to precursors of PG (arachidonic acid and eicosapentaenoic acid) on the content of
proitaglandins in breast milk?
Z>. Koldovsky: There have been some attempts to influence the amount of PG in
mill by diet, but as far as I know the results are negative. Even with drastic alteration
in tie lipid content of the milk, the levels of PG were unchanged.
Z>. Ogra: We have recently looked at leukotrienes and thromboxanes in milk, and
I beieve that most of these products come from neutrophils in the milk. If a relatively
higr content of neutrophils is found in your milk sample, it is quite possible to have
quit high concentrations of thromboxanes and, presumably, also prostaglandins.
Z>. Raibaud: Do you know if the metabolism of EGF has been studied in germfree rodents? As you know, the stomach of rodents is normally populated with lactobjcilli and micrococci, even on the gastric wall. Might these not influence your
results?
Z>. Koldovsky: I do not think so. EGF is not degraded very much in the stomach;
ovc 90% of it survives gastric passage. The other part of the gastrointestinal tract
where EGF might be metabolized is the colon, but I think it survives there too—it
is sich a small polypeptide, pepsin resistant, trypsin resistant, HC1 resistant. It is
an ideal molecule to survive every insult!