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Nutritional Science Laboratory and * Biochemical Research Laboratory, Morinaga Milk Industry Company, Zama, Kanagawa 228-8583, Japan
2To whom correspondence should be addressed. E-mail: jcb00322{at}nifty.ne.jp.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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200, 20 and 1
µmol/L, respectively. These data indicate that
functional fragments of LF such as fragments containing
glycosaminoglycan-binding site(s), as well as large fragments with
a mass >20 kDa, indeed survive proteolytic degradation in the small
intestine of adult rats.
KEY WORDS: • lactoferrin • lactoferricin • digestion • SELDI affinity mass spectrometry • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,2)
. LF is also stored in
granules of neutrophils and is a major protein component of secreted
fluids such as saliva, gastric juice and bile (3)
.
Specific LF receptors have been found on intestinal brush border
(4
,5)
, hepatocytes (6)
and peripheral blood
cells (7)
, although their functions are not yet
well characterized. It is generally recognized that orally administered
proteins are hydrolyzed rapidly in the gastrointestinal tract and thus
may lose any biological activity other than a nutritional function.
Early reports concerning the proteolytic degradation of LF in vitro
showed only comparative stability. However, an antimicrobial peptide
more potent than LF, even when compared in equimolar amounts, was found
after hydrolysis of LF by pepsin. This peptide has been purified and
characterized, and it is called lactoferricin (LFcin) (8)
.
The natural occurrence of LFcin, generated from ingested LF in the
stomach, has also been documented (9)
. It now appears that
the LFcin region of LF is not only the antimicrobial active center of
LF, but it may also play an important role in the interactions between
LF and its receptor on lymphocytes (7)
, between LF and
glycosaminoglycan (10
,11)
and between LF and
lipopolysaccharide (12)
.
Because LF was originally found to be a stable protein and because it
is thought to be a component functioning as a nonspecific
immunopotentiator in breast milk, infant formula containing native LF,
purified on an industrial scale, has been on the market since the
1980s. Because of incomplete development of the digestive system of
infants, ingested LF is not highly degraded in the gastrointestinal
tract (13
,14)
and absorption of nearly intact LF molecules
occurs in preterm infants (15)
. Lately, pharmaceutical and
food-related applications of LF in adults, such as its use in
treatment of patients with chronic hepatitis C (16)
, its
use as a chemopreventor of carcinogenesis (17
,18)
and as
an immunopotentiator in patients with leukemia receiving chemotherapy
(19)
have attracted considerable attention. However, very
little information is available on the metabolic fate of ingested LF in
adults. There have been extremely few reports on the fate of intrinsic
LF secreted into the gastrointestinal tract of adults or on the fate of
administered LF. This could be due to lack of a suitable means of
detecting protein fragments present in trace amounts during transit
through the digestive system of adults. ELISA, Western blotting, and
immunohistochemistry are the methods most commonly used for detecting
proteins. Some controversy exists concerning whether these
immunological methods are suitable for the precise characterization of
LF fragments, or for monitoring the occurrence of antimicrobial
fragments of LF, due to possible cross-reactivity with inactive
highly degraded fragments and cross-reactivity with endogenous LF.
The main focus of the present study was to characterize the digestion
of LF in adult rats with accurate identification of LF and its
fragments.
Recently, we developed a new method for detection of LF and its
fragments, involving in situ hydrolysis and surface-enhanced laser
desorption/ionization (SELDI) mass spectrometry (9)
. This
method can detect multiple forms of degraded LF as simple molecular ion
peaks corresponding to one of the core regions of LF, namely, LFcin.
Unlike the conventional methods previously used in attempts to
characterize the process of digestion of LF, the SELDI affinity mass
spectrometric assay can distinguish a certain group of LF fragments
(i.e., fragments containing the LFcin region) from various contaminants
by means of both the peptide map obtained upon in situ hydrolysis and
the precise mass values obtained through the coupled mass spectrometric
analysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male F344Crj rats (9 wk old) were obtained from Charles River Japan
(Kanagawa, Japan). They were initially allowed free access to a
commercial pelleted diet
(MF:4
oriental Yeast, Tokyo, Japan) and tap water. Neither intact LF nor LF
fragments were detected in the diet upon examination by SELDI affinity
mass spectrometry (9)
. This study was performed in
accordance with the NIH guidelines (20)
.
Materials.
Because commercially prepared bovine LF shows batch-to-batch variation
in terms of iron saturation and because its susceptibility to enzymatic
digestion is influenced by the degree of iron saturation, lyophilized
LF from Morinaga Milk Industry (Tokyo, Japan) with an iron saturation
level of 15–20% was used in all experiments. Bovine LFcin (MW 3123.8,
3194.9) was purified by the procedure described previously
(8)
, and its amino acid sequence and molecular mass were
confirmed to be the same as reported previously (8
,21)
.
The 21-residue histidine-rich glycoprotein 4-mer (HRG 4-mer, MW
2337.4), used as an internal standard peptide for mass spectrometry
(22)
, was chemically synthesized and obtained from Peptide
Institute (Osaka, Japan). Matrix-assisted laser
desorption/ionization (MALDI) and SELDI mass spectrometry were
performed using a ProteinChip Reader Model PBS I or II system
(Ciphergen, Fremont, CA), which is a linear time-of-flight mass
spectrometer with a 0.8-m flight pathlength. Laser pulses (4 ns) were
generated from a nitrogen laser (337 nm). Data were captured at 250 or
500 megasamples/s with a digital oscilloscope and were analyzed with
PC-based software. The spectra presented here typically represent
the average of 10–30 shots unless otherwise stated.
Radiolabeling of LF.
LF was labeled with 125I by the Iodogen method
(23)
. In each of five conical polypropylene vials coated
with Iodo-Gen (Pierce, Rockford, IL), 100 µg of LF
in 40 µL of water was mixed with 33.3 MBq of
[125I] NaI (Amersham Pharmacia Biotech, Uppsala, Sweden)
and allowed to react for 1 min at room temperature and at pH 8. The
unbound iodine was removed by gel filtration on a PD-10 column
(Amersham Pharmacia Biotech). The radiolabeled fraction was 98.8%
trichloroacetic acid precipitable and the specific activity of
125I-labeled LF was 233 kBq/µg. The amount
of labeled iodine was calculated as an average of one iodine atom per
3.8 molecules of LF (i.e., 0.26 iodine atoms per LF molecule). Because
at least 4 of the 21 tyrosine residues of LF (PIR database entry TFBOL, sequence revision 21 November, 1997) play an important role in both the
iron-binding activity of LF and its conformation, the properties of
iodinated LF were checked carefully. Radiolabeled LF was analyzed by
HPLC using a Cosmosil 5-Diol 300 column (7.5 x 300 mm, Nacalai
Tesque, Tokyo, Japan), and the percentage of radioactivity associated
with LF was 90.4%. No difference in the elution pattern or the
retention time was noted by HPLC analysis comparing unlabeled LF and
125I-labeled LF. The total iron-binding capacity of
127I-labeled LF was 83.4% of the original level when
tested under the same labeling conditions. The total iron-binding
capacity is the sum of the bound iron and the unbound iron-binding
capacity. The bound iron and the unbound iron-binding capacity were
determined by means of commercial kits, FeB-test WAKO and
UIBC-test WAKO (Wako Pure Chemical, Osaka, Japan), respectively.
Electrophoretic analysis of LF fragments in the gastrointestinal tract.
Taking into account the pharmaceutical and food-related
applications, the concentration of LF in the solution administered in
this study was the same or slightly higher than that in human colostrum
(60–361 µmol/L, 5–30 mg/mL) (24)
, which
would allow a sufficient amount of LF to survive digestion in the
gastrointestinal tract when administered orally to human adults. The
125I-labeled LF solution was prepared by mixing 3.3 mL of
125I-labeled LF solution (106.4 MBq) and 1.65 mL of a 200
mg/mL solution of unlabeled LF. The rats were deprived of food for
18 h before ingestion of LF. Rats weighing 200–220 g were
administered a solution containing 64.2 Bq of labeled LF/200 mg of
unlabeled LF/(3 mL · kg) by gavage. At 20, 60, 180, 360 and 720 min
postingestion, one rat each was anesthetized with ether. In a control
experiment, [125I] NaI (67.5 MBq/kg) was mixed with
unlabeled LF and administered in the same manner and the rat was
anesthetized with ether at 60 min postingestion. The small intestine
was excised and divided into two segments of equal length. Each segment
was washed twice with 5 mL of ice-cold PBS containing protease
inhibitors (Complete protease inhibitors: Boehringer Mannheim,
Mannheim, Germany; Pefabloc SC: Boehringer Mannheim; Pepstatin A: Sigma
Chemical, St. Louis, MO). The segments of the intestinal tract before
and after washing were weighed and the weight of the inner contents was
calculated by subtraction. Because the density of the contents was 1
kg/L, the concentration of LF fragments in the contents was determined
per milliliter instead of per gram. The recovered contents were
homogenized using a Polytron homogenizer (Kinematica, Lucerne,
Switzerland) and were frozen at -80°C before use. The compounds
labeled with 125I were separated by SDS-PAGE. The dried
gel was exposed to an imaging plate in a cassette, and the exposed
plate was analyzed using a Fujix BAS 2000 radioluminography system. The
amount of radioactivity associated with each band was determined from
the photo-stimulated luminescence (PSL) by mathematical
integration. Assuming that one LF molecule contained one
125I atom or less, the amount of LF fragments in the
intestinal contents was calculated by comparison of the PSL of each
PAGE band with that of a known amount of standard
125I-labeled LF.
Quantitative determination of LF fragments in the small intestine of rats after a single dose of LF.
Rats were given a solution containing 200 mg of LF/(3 mL · kg) or
the vehicle (water; 3 mL/kg) by gavage. The rats were anesthetized with
ether at 60 min postingestion and the small intestine was excised.
The lower segment of the small intestine was washed twice with 5 mL of
PBS. The recovered contents were homogenized and the weight of the
contents was calculated as mentioned above. LF fragments containing the
LFcin region (i.e., Phe17-Ala42) were quantified by SELDI affinity mass
spectrometry specific for LF fragments as described previously [see
Methods 2.2 and the flow chart in Fig. 2
of reference
(9)
]. Briefly, a portion of the recovered suspension
(0.1–10 mL) was mixed with 2 volumes of extraction buffer containing 1
mol/L NaCl and 9 mol/L urea, followed by continuous mixing by vortex at
4°C and then the sample was centrifuged. The supernatant was
recovered and diluted 2- to 4000-fold with 6 mol/L urea. To a portion
of the diluted extract (1.0–60 mL), a 50% (v/v) suspension of Butyl
Toyopearl 650M (Tosohaas, Montgomeryville, PA) was added, followed by
mixing at 4°C. All solutions up to this stage contained the protease
inhibitors mentioned above. LF and its fragments captured by the gel
were hydrolyzed by pepsin, recaptured and analyzed by laser
desorption/ionization time-of-flight mass spectrometry. Various LF
fragments containing the LFcin region were hydrolyzed and then were
detected as molecular ion peaks corresponding to LFcin. The mass
spectrum was normalized by the peak intensity of a known amount of the
internal standard peptide (HRG 4-mer), which was added as a component
of a solution of 
-cyano-4-hydroxycinnamic acid at the
cocrystallization step. The use of the internal standard in
quantitative mass spectrometric analysis has been described previously
(25)
. The amount of LFcin recaptured after in situ
hydrolysis was calculated from the normalized peak intensity of the
molecular ion peaks of LFcin. The corresponding amount of LF in the
contents of the gastrointestinal tract was calculated from the
estimated amount of LFcin, based on the molecular mass ratio [3.2 kDa
(LFcin)/83 kDa (LF)]. The amount of LF was expressed as the mean value
for three rats.
|
Rats were divided randomly into two groups of three rats each. One
group was fed commercial bovine milk (Morinaga Milk Industry) and the
other group was fed milk enriched with bovine LF at a concentration of
482 µmol/L (40 mg/mL). The ordinary milk or milk
enriched with LF was consumed ad libitum using a sterile nozzle (AN
pack and SE nozzle, Musashi, Saitama, Japan). Rats were fed the liquid
diets as the sole source of food. Their milk consumption was roughly
estimated to be 60 mL/(rat · d) and the LF intake was calculated to
be 2.4 g/(rat · d). The sterile bags containing each liquid diet
were changed every 2 d. After 1 wk of feeding, the rats were
anesthetized with pentobarbital and the gastrointestinal tract was
excised. LF and its fragments in the contents of the gastrointestinal
tract were quantified by SELDI affinity mass spectrometry in the same
manner as described above. The amount of LF was expressed as the mean
value for three rats. The recovery of standard LF added to the contents
of the gastrointestinal tract was within the range of 63–85%.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the degradation pattern of ingested LF, rats were orally
administered 125I-labeled LF solution comprising
a dose of 64.2 MBq in 200 mg of LF/(3 mL · kg). Molecular forms of
125I-labeled LF in the contents of the upper and
lower small intestine in the period from 20 to 720 min postingestion
were analyzed by SDS-PAGE and autoradioluminography (Fig. 1
). Radioactivity attributable to intact LF was not found in the small
intestine at any sampling time (Fig. 1c
, lanes 1–9).
Radioactive fragments having molecular masses of 42, 36, 33, 29, 15 and
5 kDa were evident in the contents of the upper small intestine at 20
min postingestion (Fig. 1c
, lane 1). At 60 min
postingestion, no radioactive fragments were seen in the contents of
the upper small intestine (Fig. 1c
, lane 2). In the lower
small intestine, however, radioactive fragments having molecular masses
of 42, 36, 33, 29, 15 and 5 kDa were evident at 20, 60 and even 180 min
postingestion (Fig. 1c
, lanes 4–6). The amount of
radioactive fragments with a relatively high molecular mass, such as
molecular masses of 42 and 29 kDa, reached a maximum at 60 min
postingestion (Fig. 1c
, lane 5) but these fragments
disappeared at 360 min postingestion or later (Fig. 1c
, lane
7 and 8). The amount of the 5- and 3-kDa radioactive fragments had
increased at 180 min (Fig. 1c
, lane 6), then had decreased
at 360 min (Fig. 1c
, lane 7) and had mostly disappeared at
720 min postingestion (Fig. 1c
lane 8). In the control
experiment (i.e., administration of [125I] NaI
instead of 125I-labeled LF), low but relevant
amounts of radioactivity attributable to fragments with a mass of 5 kDa
in the upper small intestine, and 10, 5 and 3 kDa in the lower small
intestine, were detected at 60 min postingestion (Fig. 1c
,
lanes 3, 9). The radioactivity in the gel seemed to be derived from
free 125I nonspecifically interacting with
components of the intestinal contents. Thus, the radioactive
constituents with a molecular mass >20 kDa could be fragments of
ingested 125I-labeled LF (Fig. 1c
,
lanes 1, 4, 5 and 6; positions indicated by arrows). Comparing the
radioactivity of the bands with that observed in the case of standard
125I-labeled LF (Fig. 1b
), the amount
of LF fragments with a mass >20 kDa in the contents was approximated
and the results were as follows: 4.7 mg as LF equivalents/mL (56.6
µmol/L) in the upper small intestine at 20 min, 28.3
µg as LF equivalents/mL (341 nmol/L), 29.7 µg
as LF equivalents/mL (358 nmol/L), and 8.9 µg as LF
equivalents/mL (107 nmol/L) in the lower small intestine at 20, 60 and
180 min. The percentage of recovery of radioactivity associated with
fragments having a mass >20 kDa in the washing fluid collected from
the upper small intestine at 20 min postingestion was 2.6% of the
total administered dose of radioactivity, and that in the case of the
washing fluid collected from the lower small intestine was 0.014, 0.036
and 0.005% at 20, 60 and 180 min postingestion, respectively. It
should be noted, however, that radioactive constituents remained at the
top edge of the gel (Fig. 1c
, lane 6 and 7) and this hinders
any further precise quantitation of LF fragments.
|
Using 125I-labeled LF, some controversy still
remained concerning the precise identification and quantitation of LF
fragments even though a control experiment was employed. Mass
spectrometric analysis providing an accurate mass estimate (typically
<± 0.05%) of a peptide mapped to a certain region of LF (i.e.,
LFcin) offers advantages of precise characterization and quantitative
determination of the digestive fate of ingested LF. Figure 2
shows the results obtained in the SELDI affinity mass spectrometric
assay for LF fragments in the contents of the lower small intestine of
rats fed a solution containing 200 mg of LF/(3 mL · kg)
(upper spectrum) or the vehicle (middle spectrum)
at 60 min postingestion, and the MALDI mass spectrum of standard LFcin
(lower spectrum). Clearly, molecular ion peaks of
constituents with a mass identical to standard LFcin were evident in
the spectrum obtained from the contents of the lower small intestine of
rats fed LF (Fig. 2
, upper spectrum), whereas no molecular
ion peaks that could be assigned to LFcin were found in the case of
rats fed the vehicle (Fig. 2
, middle spectrum). The amount
of LFcin was calculated from the peak intensity of the normalized
molecular ion peaks. The amount of LF fragments containing the LFcin
region in the contents of the lower small intestine was 30 nmol/L (2.5
µg as LF equivalents/mL).
Quantitative determination of LF in the gastrointestinal tract of rats fed a diet supplemented with LF.
To characterize the digestive fate of LF ingested as a dietary
supplement, the contents of the stomach, upper small intestine and
lower small intestine were analyzed by SELDI affinity mass spectrometry
(Fig. 3
). Rats were fed milk enriched with LF at a concentration of 482
µmol/L (40 mg/mL). Molecular ion peaks corresponding to
LFcin were evident in the spectrum obtained on analysis of the gastric
contents (Fig. 3a
). Compared with the gastric contents, the
contents of the upper small intestine showed a LFcin signal of lower
intensity (Fig. 3a
, b
). This could indicate that
degradation of ingested LF into oligopeptides such as fragments smaller
than LFcin and/or absorption of LF fragments had occurred in the small
intestine. Thereafter, however, molecular ion peaks corresponding to
LFcin were still observed on analysis of the contents of the lower
small intestine (Fig. 3c
). The amount of LF fragments
detected in the contents of the stomach, upper small intestine and
lower small intestine was approximately 200 µmol/L (17 mg
as LF equivalents/mL), 20 µmol/L (1.7 mg as LF
equivalents/mL) and 1 µmol/L (83 µg as LF
equivalents/mL), respectively. In contrast, no molecular ion peaks that
could be assigned to LFcin were found in analysis of the gastric
contents of control rats fed milk alone (Fig. 3d
). Standard
LF added at a concentration of 482 µmol/L (40 mg/mL) to
the gastric contents of control rats was detected as molecular ion
peaks corresponding to LFcin (Fig. 3e
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,26)
but also in
receptor studies (27
,28)
. Many investigations examining
the fate of other protein/peptide drugs applicable to humans using a
rat model have permitted some insight to be obtained.
This is the first report on the quantitative analysis of ingested LF in
the gastrointestinal tract with accurate identification of the protein
and its fragments. Various problems with immunological detection of LF
have been reported previously, including possible cross-reactivity
with inactive fragments from ingested LF, endogenous LF and transferrin
(9
,29)
Figure 4
shows a summary of the results of quantitative determination of LF in
this study. The LF concentration in the gastrointestinal tract under
different conditions was roughly determined using 1–3 rat(s) and was
plotted on a logarithmic scale together with intrinsic LF levels in
humans (24
,30
,31)
. We employed two animal models of human
oral intake, one involving administration of a single dose and the
other involving dietary supplementation (Fig. 4)
. In both cases, the
concentration of LF fragments in the contents of the upper small
intestine remained higher than the minimum inhibitory concentration of
LFcin against some sensitive bacteria (8)
(Fig. 4)
. Even
after degradation and/or absorption in the small intestine, LF
fragments were detected at a concentration of at least 10 nmol/L (830
ng as LF equivalents/mL) in the contents of the lower small intestine
(Fig. 4)
. This concentration is comparable to that at which some
physiologic effects of LF are displayed in vitro, such as its effects
on cell proliferation (32)
and cytokine production
(33)
.
|
. In contrast, we demonstrated that LF fragments could
survive transit through the small intestine in adult rats in
substantial amounts (10–1000 µmol/L) (Fig. 4)
. The
discrepancy with respect to the stability of LF against digestion in
adult animals may be due mainly to the difference in animals tested,
the difference in sensitivity of the detection methods used and the
difference in protease inhibitors used during recovery of the
gastrointestinal contents.
After administration of a single dose of LF, although the conditions of
administration were similar, the concentration of LF fragments in the
contents of the lower small intestine at 60 min postingestion as
determined by SELDI affinity mass spectrometry (30 nmol/L) and that
determined by radiolabeling (358 nmol/L) differed by nearly one order
of magnitude (Fig. 4)
. One possible reason is adsorption of certain LF
fragments to cells in the small intestine. The
glycosaminoglycan-binding site of LF overlaps with the region
quantified by SELDI affinity mass spectrometry (i.e., Phe17-Ala42)
(10
,11)
. Glycosaminoglycans occur on the surface of most
cells and in matrices. They often bind biologically active molecules,
sequester them and present them to specific receptors. The
glycosaminoglycan-binding region of LF is not detectable by the
125I-labeling method because the N-terminal
region of LF (i.e., the region Ala1-Pro71) contains no tyrosine
residues (PIR database entry TFBOL, sequence revision 21 November 1997), which serve as the main target of iodination. In addition to
glycosaminoglycan-mediated binding, a specific LF receptor in the
intestinal brush border has been reported (4
,5)
. Kawakami
et al. (27)
reported the specific binding of bovine LF to
rat brush border membranes. The LFcin region of LF may play an
important role in the interaction between LF and its receptor on
lymphocytes (7)
. Although the region of LF essential
for intestinal receptor binding is not known, LF fragments containing
the LFcin region are likely to bind with the intestinal receptor. The
possibility of adsorption of certain LF fragments to cells in the small
intestine through nonspecific interactions (i.e.,
glycosaminoglycan-mediated binding) and/or specific interactions
(i.e., receptor-mediated binding) should be elucidated by
performing further studies using brush border membrane vesicles and
mutant forms of LF with the N-terminal region deleted.
Brock et al. (35)
reported that when bovine LF was
hydrolyzed by trypsin in vitro, fragments with molecular masses of
52.7, 47.3 and 31.8 kDa were produced and these fragments showed
resistance to further degradation. In the present study, we also found
such relatively large fragments in the contents of the lower small
intestine even at 180 min postingestion (Fig. 1)
. Taking into account
the error in mass estimates obtained by gel electrophoresis (±15%),
the stable LF fragments found in our study with masses of 42 and 29 kDa
could be the same fragments as those reported by Brock et al.
(35)
The structural relationship responsible for the
observation that tryptic fragments of LF with a relatively high mass,
generated in the small intestine, showed unusual resistance to further
proteolytic degradation is worthy of further study. The stable LF
fragments with a mass of >20 kDa disappeared from the lower small
intestine at 360 min or later (Fig. 1)
. In the contents of the cecum
and colon, such radioactive fragments were not detected at 180, 360 and
720 min postingestion (data not shown). Thus, the stable LF fragments
observed in the lower small intestine at 60 and 180 min postingestion
may have been degraded further and/or absorbed into the body.
Many studies have examined the stability and absorption of ingested LF
in infants. In our study using adult rats, a substantial amount of LF
fragments survived proteolytic degradation in the small intestine. The
amount of relatively large fragments derived from ingested LF reached a
maximum in the lower small intestine at 60 min postingestion. At around
the same time point, judging from the results of whole-body
autoradiography performed under the same conditions, the speed of
absorption of radioactivity from the gut and the distribution of
radioactivity throughout the body had also reached a maximum
(36)
. It is possible that not only highly degraded LF
(e.g., oligopeptides, amino acids) but also relatively large fragments
could be absorbed through the gut wall and be distributed to some
organs such as the liver at this time. Circulation of LF from the
intestinal lumen into the bile via the blood in nursing miniature pigs
has been reported (37)
. In general, however, the degree of
absorption of relatively large protein fragments in the small intestine
of adult animals is very limited (38
,39)
. Intestinal
absorption of oligopeptide drugs composed of D-amino acids,
which are resistant to proteolytic degradation, is reported to occur
only when junctional permeability is increased by
Na+-coupled transport of glucose
(40)
. A preliminary experiment using SELDI affinity mass
spectrometry (data not shown) indicated that the concentration of
absorbed LF fragments in the portal blood of an adult rat at 60 min
postingestion of 200 mg of LF/(3 mL · kg) was < 100 pmol/L,
(8.3 ng/mL). Supposing that there is minimal absorption of relatively
high mass fragments of LF, interactions between the LF
fragments that have survived digestion and cells in the intestinal
tract could have some relevance in terms of the biological functions of
this protein. Further studies are required to clarify the receptor
binding, the extent of absorption of LF fragments through the gut wall,
the levels of these fragments in the circulation, their tissue
distribution and their relation to the biological functions of LF.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: HRG, histidine-rich glycoprotein; LF, lactoferrin; LFcin, lactoferricin; MALDI, matrix-assisted laser desorption/ionization; PSL, photostimulated luminescence; SELDI, surface-enhanced laser desorption/ionization.
4 Standard nonpurified MF diet (Oriental Yeast) contains the following nutrients per kg: water, 7.6 g; protein, 246 g; lipid 56 g; carbohydrate 528 g; minerals (41)
, 63 g; vitamins (41)
, 7 g.
Manuscript received December 27, 2000. Initial review completed February 11, 2001. Revision accepted May 11, 2001.
|
REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1.
S
rensen M., Srensen S.P.L. The proteins in whey. C. R. Trav. Lab. Carlsberg 1939;23:55-99
2. Masson P. L., Heremans J. F. Lactoferrin in milk from different species. Comp. Biochem. Physiol. B 1971;39:119-129[Medline]
3. Masson P. L., Heremans J. F., Dive C. H. An iron binding protein common to many external secretions. Chin. Chim. Acta 1966;14:735-739
4.
Davidson L. A., Lönnerdal B. Specific binding of lactoferrin to brush-border membrane: ontogeny and effect of glycan chain. Am. J. Physiol. 1988;254:G580-G585
5. Mazurier J., Montreuil J., Spik G. Visualization of lactotransferrin brush-border receptors by ligand-blotting. Biochim. Biophys. Acta 1985;821:453-460[Medline]
6.
McAbee D. D., Esbensen K. Binding and endocytosis of apo- and holo-lactoferrin by isolated rat hepatocytes. J. Biol. Chem. 1991;266:23624-23631
7. Legrand D., Mazurier J., Elass A., Rochard E., Vergoten G., Maes P., Montreuil J., Spik G. Molecular interactions between human lactotransferrin and the phytohemagglutinin-activated human lymphocyte lactotransferrin receptor lie in two loop-containing regions of the N-terminal domain I of human lactotransferrin. Biochemistry 1992;31:9243-9251[Medline]
8. Bellamy W., Takase M., Yamauchi K., Wakabayashi H., Kawase K., Tomita M. Identification of the bactericidal domain of lactoferrin. Biochim. Biophys. Acta 1992;1121:130-136[Medline]
9. Kuwata H., Yip T. T., Tomita M., Hutchens T. W. Direct evidence of the generation in human stomach of an antimicrobial peptide domain (lactoferricin) from ingested lactoferrin. Biochim. Biophys. Acta 1998;1429:129-141[Medline]
10.
Mann D. M., Romm E., Migliorini M. Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein. J. Biol. Chem. 1994;269:23661-23667
11. Shimazaki K., Tazume T., Uji K., Tanaka M., Kumura H., Mikawa K., Shimo-Oka T. Properties of a heparin-binding peptide derived from bovine lactoferrin. J. Dairy Sci. 1998;81:2841-2849[Abstract]
12. Elass-Rochard E., Roseanu A., Legrand D., Trif M., Salmon V., Motas C., Montreuil J., Spik G. Lactoferrin-lipopolysaccharide interaction: involvement of the 28–34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide. Biochem. J. 1995;312:839-845
13.
Britton J. R., Koldovsky O. Luminal digestion of lactoferrin in suckling and weanling rats. Am. J. Physiol. 1987;253:G397-G403
14. Spik G., Brunet B., Mazurier-Dehaine C., Fontaine G., Montreuil J. Characterization and properties of the human and bovine lactotransferrins extracted from the faeces of newborn infants. Acta Paediatr. Scand. 1982;71:979-985[Medline]
15.
Hutchens T. W., Henry J. F., Yip T. T. Structurally intact (78-kDa) forms of maternal lactoferrin purified from urine of preterm infants fed human milk: identification of a trypsin-like proteolytic cleavage event in vivo that does not result in fragment dissociation. Proc. Natl. Acad. Sci. U.S.A. 1991;88:2994-2998
16. Tanaka K., Ikeda M., Nozaki A., Kato N., Tsuda H., Saito S., Sekihara H. Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C: a pilot study. Jpn. J. Cancer Res. 1999;90:367-371[Medline]
17. Sekine K., Watanabe E., Nakamura J., Takasuka N., Kim D. J., Asamoto M., Krutovskikh V., Baba-Toriyama H., Ota T., Moore M. A., Masuda M., Sugimoto H., Nishino H., Kakizoe T., Tsuda H. Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats. Jpn. J. Cancer Res. 1997;88:523-526[Medline]
18. Yoo Y. C., Watanabe S., Watanabe R., Hata K., Shimazaki K., Azuma I. Bovine lactoferrin and lactoferricin, a peptide derived from bovine lactoferrin, inhibit tumor metastasis in mice. Jpn. J. Cancer Res. 1997;88:184-190[Medline]
19. Trumpler U., Straub P. W., Rosenmund A. Antibacterial prophylaxis with lactoferrin in neutropenic patients. Eur. J. Clin. Microbiol. Infect. Dis. 1989;8:310-313[Medline]
20. National Research Council Guide for the Care and Use of Laboratory Animals 1985 National Institutes of Health Washington, DC. Publication no. 85–23(rev.)
21. Kuwata H., Yip T. T., Yip C. L., Tomita M., Hutchens T. W. Bactericidal domain of lactoferrin: detection, quantitation, and characterization of lactoferricin in serum by SELDI affinity mass spectrometry. Biochem. Biophys. Res. Commun. 1998;245:764-773[Medline]
22. Hutchens T. W., Nelson R. W., Allen M. H., Li C. M., Yip T. T. Peptide-metal ion interactions in solution: detection by laser desorption time-of-flight mass spectrometry and electrospray ionization mass spectrometry. Biol. Mass Spectrom. 1992;21:151-159
23. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem. Biophys. Res. Commun. 1978;80:849-857[Medline]
24. Kawakata N. The isolation and characterization of human milk lactoferrin and the fluctuations of its concentration at different stages of lactation. Nippon Ika Daigaku Zasshi 1984;51:426-440[Medline]
25. Nelson R. W., McLean M. A., Hutchens T. W. Quantitative determination of proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 1994;66:1408-1415
26.
Regoeczi E., Chindemi P. A., Debanne M. T., Prieels J. P. Lactoferrin catabolism in the rat liver. Am. J. Physiol. 1985;248:G8-G14
27.
Kawakami H., Dosako S., Lönnerdal B. Iron uptake from transferrin and lactoferrin by rat intestinal brush-border membrane vesicles. Am. J. Physiol. 1990;258:G535-G413
28. McAbee D. D., Jiang X., Walsh K. B. Lactoferrin binding to the rat asialoglycoprotein receptor requires the receptor’s lectin properties. Biochem. J. 2000;348:113-117
29. Magnuson J. S., Henry J. F., Yip T. T., Hutchens T. W. Structural homology of human, bovine, and porcine milk lactoferrins: evidence for shared antigenic determinants. Pediatr. Res. 1990;:176-181
30. Masson P. L., Heremans J. F., Dive C. H. An iron-binding protein common to many external secretions. Clin. Chim. Acta 1966;14:735-739
31.
Levay P. F., Viljoen M. Lactoferrin: a general review. Haematologica 1995;80:252-267
32. Nichols B. L., McKee K. S., Henry J. F., Putman M. Human lactoferrin stimulates thymidine incorporation into DNA of rat crypt cells. Pediatr. Res. 1987;21:563-567[Medline]
33.
Crouch S. P., Slater K. J., Fletcher J. Regulation of cytokine release from mononuclear cells by the iron-binding protein lactoferrin. Blood 1992;80:235-240
34.
Drescher K., Roos N., Pfeuffer M., Seyfert H. M., Schrezenmeir J., Hagemeister H. Recovery of 15N-lactoferrin is higher than that of 15N-casein in the small intestine of suckling, but not adult miniature pigs. J. Nutr. 1999;129:1026-1030
35. Brock J. H., Arzabe F., Lampreave F., Pineiro A. The effect of trypsin on bovine transferrin and lactoferrin. Biochim. Biophys. Acta 1976;446:214-225[Medline]
36. Kuwata H., Ushida Y., Shimokawa Y., Toida T., Yamauchi K., Teraguchi T., Hayasawa H., Shimamura S., Tomita M. Digestion of orally administered lactoferrin in adult rats. Shimazaki K. Tsuda H. Tomita M. Kuwata T. Perraudin J. P. eds. International Congress Series 1195. Lactoferrin Structure, Function and Applications 2000:311-317 Elsevier Science B. V. Amsterdam, The Netherlands.
37. Harada E., Itoh Y., Sitizyo K., Takeuchi T., Araki Y., Kitagawa H. Characteristic transport of lactoferrin from the intestinal lumen into the bile via the blood in piglets. Comp. Biochem. Physiol. A 1999;124:321-327
38.
Matthews D. M. Intestinal absorption of peptides. Physiol. Rev. 1975;55:537-608
39. Matthews D. M. Intestinal absorption of peptides. Biochem. Soc. Trans. 1983;11:808-810[Medline]
40.
Pappenheimer J. R., Dahl C. E., Karnovsky M. L., Maggio J. E. Intestinal absorption and excretion of octapeptides composed of D amino acids. Proc. Natl. Acad. Sci. U.S.A. 1994;91:1942-1945
41. American Institute of Nutrition Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 1977;107:1340-1348
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