Martha,
This might be helpful. It is a patent called "Lactoferrin as a dietary
ingredient promoting the growth of the gastrointestinal tract." This patent
is for infant formula but what they say about the necessity of human milk for
the health of the newborn is worth reading. This is patent # 4,977,137 and
Baylor College of Medicine is the assignee to this patent (Baylor does alot
of human milk studies). Anyone can access this patent by going to the US
Patent Office web site:
http://www.uspto.gov/patft/index.html then do a number search--type in
patent #
sorry for the length, Valerie W. McClain, IBCLC
BACKGROUND OF THE INVENTION
A substantial growth of the intestines of newborn animals takes place in the
first one to three days after birth. For example, in newborn pigs who are
nursed by the mother, there is a substantial growth, approximately eight to
ten inches, of the intestines of the infant within the first few days after
birth. In a large number of human newborns, who are not nursed by the mother
but are placed on an infant's formula, this growth of the gastrointestinal
tract during the first few days may not occur, and, as a result, the infant
is predisposed to chronic intractable diarrhea which must be managed for a
period of three or more months at considerable expense and discomfort to the
infant.
The present invention is based upon the discovery that milk lactoferrin as a
dietary ingredient promotes the growth of the gastrointestinal tract when
added to infant formula or given separately from the formula and thus reduces
the occurrence of chronic diarrhea and may assist in the management of
short-gut syndrome and avoids, at least to some extent, chronic intractable
diarrhea of the infant. The lactoferrin may be from a nonhuman animal or
human source. The milk containing the lactoferrin should not be processed,
such as by pasturization or the lactoferrin processed, extracted, or purified
by a process which destroys the effectiveness of the lactoferrin.
Mammary secretions from goats, sheep, cows, and humans have been found to
stimulate the proliferation of various cell lines growing in culture (1-3).
When purified, the active factor was found to be Epidermal Growth Factor
(EGF). None of the cell lines used for the bioassay of previous mitogenic
factors exhibit polarity and none have brush border membranes.
In a search for factors in human colostrum which might stimulate enterocyte
proliferation, we developed an assay for thymidine incorporation into DNA
using harvested crypt cells from mature rat small intestine. Whole skimmed
human colostrum stimulated a significant increase in thymidine incorporation
into crypt cell DNA during a 60 minute period of incubation. When the protein
with biological activity was purified to a single peak by sequential ion and
gel chromatography, it was found to have the characteristics of lactoferrin.
The mobility of SDS electrophoresis and electrofocusing was identical to that
of standard lactoferrin. The protein was identical to these standards on
double-diffusion immunologic testing. All available human lactoferrins
stimulated thymidine uptake and all reacted with a lactoferrin polyclonal
antibody. Human lactoferrin appears to be a potent activator of thymidine
incorporation into DNA in incubated rat crypt cells, a biological activity
not previously reported.
In addition, we found that EGF does not stimulate crypt cells. This finding
supports our hypothesis that the response of 3T3 fibroblasts to EGF is not
synonymous with a trophic effect on polarized intestinal epithelial cells.
Studies in the pig, dog, and rat indicate that the gastrointestinal tract
matures more rapidly if the newborn animal is suckled (1-3). The nutritional
significance of these observations lies in the principle that the structure
of ingested protein may have biological significance beyond the dietary
requirement for amino acids. Based on these observations, in vitro
fibroblasts and other cell lines have been used to test for the presence of
growth-promoting factors in milk. Mammary secretions from goats, sheep, cows,
and humans have been found to stimulate the proliferation of various cell
lines growing in culture (4-6). A portion of the activity in fibroblast
culture can be attributed to EGF, a 6000-M.sub.r mitogen present in many
mammalian secretions (7). Other factors with mitogenic activity, however,
have been identified, e.g. polypeptides with M.sub.r of approximately 140,000
and 36,000(5).
None of the cell lines used for the bioassay of mitogenic factors exhibit
polarity and none have brush-border membranes. Assuming that specificity may
exist in intestinal cells, we developed a bioassay based on harvested rat
crypt enterocytes. The bioassay works equally well with harvested pig crypt
enterocytes. With this assay, we have confirmed the presence of mitogenic
activity in human colostrum. Subsequently set forth herein are the details of
the bioassay, the isolation of lactoferrin as a mitogenic dietary factor
present in human and bovine milk, and compare the enterocyte assay with the
fibroblast assay system used by Klagsbrun (5).
Although the mechanisms by which lactoferrin may stimulate the production of
DNA in crypt cells have not been described, a similar protein, transferrin,
is known to have a stimulating effect in a variety of cell lines. The two
proteins, however, are immunologically distinct. Transferrin is an essential
component of highly defined tissue culture media with a requirement of less
than 10 .mu.g/ml for most cell lines. Whether this property of transferrin is
attributable to the iron or to the apotransferrin protein has not been
determined (19). In the cell lines studied thus far, an exclusive receptor is
present for either lactoferrin or transferrin (20). Although transferrin and
lactoferrin are not interchangeable, we have discovered that lactoferrin has
a role parallel to that of transferrin in vitro and in vivo.
We have discovered that lactoferrin both human and animal stimulated
thymidine incorporation into DNA by rat or pig crypt enterocytes. Human milk
is known to stimulate thymidine uptake in a variety of fibroblast cell lines.
The factors responsible for the initiation of mitosis have been identified in
part. EGF was the first described and is the best known active factor in
human milk. Receptors at the plasma membrane of 3T3 fibroblasts bind EGF
(epidermal growth factor) and internalize it for subsequent nuclear binding.
This mechanism requires 12 to 14 hours for completion when confluent
fibroblast cultures are stimulated (16). In the assay with rat crypt cells,
the DNA was harvested after only one hour of incubation. The short incubation
may be one reason for the failure of EGF to stimulate enterocytes. The cells
in the crypt cell assay may have been conditioned in vivo by EGF before they
were harvested for the in vitro bioassay (17).
Klagsbrun and coworkers (18) have identified three factors in human milk
which stimulate cell proliferation. The 3T3 cell line responds in vitro to
whole human milk and to purified fractions. Klagsbrun's fractions I and II
and EGF accounted for 5, 20, and 75%, respectively, of the 3T3 stimulation by
human milk. Both larger fractions are broken down to smaller M.sub.r
fractions under denaturing conditions. Fraction II is resolved by isoelectric
focusing into two fractions with different pI. Based on the reported M.sub.r
and PI, none of Klagsbrun's fractions appear to be intact lactoferrin. The
cathodic protein in his factor II may be a fragment from intact lactoferrin,
but the relative resistance of lactoferrin to proteolysis makes this
possibility unlikely. Assays with 3T3 cells confirmed (data not shown) that
fractions of human colostrum stimulate thymidine incorporation in this cell
line. Human lactoferrin, however, does not promote growth in the 3T3 bioassay
which is sensitive to EGF. The absence of sensitivity to lactoferrin in the
3T3 cell line explains why previous investigators have not observed its
stimulation of thymidine incorporation in this fibroblast system.
Bovine lactoferrin purified by two different commercial processes is active
in the crypt cell bioassay. This is in contract to the lack of stimulatory
activity in cow's milk-based infant's formulas. The lactoferrin was from
bovine colostrum and bovine mature milk and was acquired from Sigma Chemical
Co., St. Louis, Mo.
The biological significance of lactoferrin-induced thymidine incorporation in
rat and pig crypt cell DNA has not been elucidated previously. What is clear,
however, is that lactoferrin is inactive in 3T3 cell lines which are
sensitive to EGF (FIG. 9) and responsive to Klagsbrun's fractions I and II
and that EGF is inactive in the crypt cell bioassay sensitive to lactoferrin
(FIG. 1).
PRIOR ART STATEMENT
A preliminary search was made of pertinent art in the U.S. Patent Office with
the following results:
U.S. Pat. No. 4,216,236 discloses a prepared infant formula from a
nutritional point of view.
Archives of Disease in Childhood, 1980, 55, 417-421 discusses lactoferrin in
human milk, its role in iron absorption and protection against enteric
infection in the newborn infant.
Chemical Abstracts, 95-113091k (1981) reviews factors of milk, including
lactoferrin which protect against intestinal infection in the newborn.
Chemical Abstracts, 104-223816n (1986) discusses preparation of fat and
protein from banked human milk and its use in feeding very low-birth-weight
infants.
The following Chemical Abstracts (CA) citations relate generally to
lactoferrin in human milk, properties of transferrin, vitamin D's in cow's
milk, infant formulas and breast milk during different stages of lactation,
the role of lactoferrin in iron absorption and its relation to nutritional
status and antimicrobial factors in whole saliva in infants.
CA 89-144306q (1978); CA 93-42320e (1980); CA 97-180804z (1982); CA
99-155959n (1983); CA 99-171500m (1983); CA 101-190192p (1984); CA 103-70198q
(1985); and CA 104-49711n (1986).
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