![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
*
Federal Dairy Research Centre, Department of Physiology and Biochemistry of Nutrition, D-24121 Kiel, Germany, and
Research Institute for Biology of Farm Animals, Division of Nutritional Physiology, D-18059 Rostock, Germany
4 To whom correspondence should be addressed.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Performance of biological functions of lactoferrin in the small intestine requires at least some resistance to degradation. Therefore, we studied prececal digestibility of lactoferrin in comparison to casein both in suckling and adult miniature pigs, applying 15N-labeled proteins. In study 1, 43 piglets (10-d-old), deprived of food for 12 h received 10 mL of sow's milk supplemented with 120 mg of 15N-labeled protein (porcine or bovine lactoferrin or bovine casein). Piglets were anesthetized 150 min later, after which the small intestine was excised, cut into three sections, and chyme was collected. In study 2, nine food-deprived boars fitted with T-canulae at the terminal ileum were given two semisynthetic experimental meals (204 g) in a cross-over design, 2 wk apart. One contained 7.5% (g/100 g) 15N-labeled bovine casein, the other 1.25% 15N-labeled bovine lactoferrin. Both were adjusted to 15% total protein with nonlabeled casein. Ileal chyme was collected from the canula over 33 h postprandially. All diets contained the indigestible marker chromic oxide. 15N-digestibility of lactoferrin, both porcine (84.4 ± 3.2%) and bovine (82.3 ± 4.8%), was significantly lower than casein digestibility (97.6 ± 0.5%) in the distal small intestine of suckling piglets (P < 0.05). Based on immunoblotting after acrylamide electrophoresis, 4.5% of non- and partially digested lactoferrin was found in the last third of the small intestine of piglets. In adult miniature pigs there was no difference in 15N-digestibility of bovine lactoferrin compared to bovine casein (90.7 ± 1.9% vs. 93.9 ± 1.0%, P > 0.05). In suckling miniature pigs, the reduced digestibility of lactoferrin may provide the prerequisite for biological actions along the whole intestinal tract. The source of lactoferrin, porcine or bovine, made no difference in this respect.
KEY WORDS: • lactoferrin • 15N • prececal digestibility • miniature pigs • suckling, adults
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionREFERENCES |
|---|
).
Lactoferrin acts as an iron carrier, shows antibacterial and
immunological effects, and has mitogenic or trophic activities on the
newborn intestinal mucosa (Hagiwara et al. 1995
,
Hambraeus 1988
, Kinsella and Whitehead 1989
). The antimicrobial activities are of particular interest.
According to Ellison and Giehl (1991)
, lactoferrin might
alter the permeability of bacterial outer membranes by three different
mechanisms. Not only the intact protein, but also pepsin-digested
or heat-damaged lactoferrins are bacteriostatic (Abe et al. 1991
, Tomita et al. 1991
). Nevertheless,
resistance against complete degradation by digestive enzymes is a
prerequisite for the biological functions of lactoferrin in the small
intestine, necessarily resulting in a low absorption. A high biological
activity of lactoferrin means an advantage for the newborn mammal
because at this age an effective defense mechanism, e.g., through a low
pH in the stomach, has not been developed (Manners 1976
).
The aim of this study was to test in miniature pigs whether there is
indeed a low digestion of lactoferrin along the whole small intestine.
Both the influences of age and of the source of lactoferrin, porcine or
bovine milk, were studied. Due to 15N-labeling of the test
proteins, it was possible to determine true digestibility (Roos et al. 1995
).
|
Materials and Methods |
|---|
|
TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionREFERENCES |
|---|
15N-labeled cow's milk (700 kg) (mean enrichment of
the dry matter 0.455 atom%) was obtained according to Roos et al. (1994)
. In short, 50 g of enriched ammonium sulfate
{([15N]H4)2SO4, 10
atom%, Medgenix, Ratingen, Germany} was diluted in 2 L of tap water
and continuously infused over 24 h into the rumen of a cow via a
permanent rumen fistula. The procedure was started 1 d before
calving and was maintained over 2 wk. Sow's milk was labeled by means
of highly 15N-enriched yeast Saccharomyces
cerevisiae (
95 atom%) and 15N-labeled cow's milk
in the following way: First 15N-enriched yeast was grown in
a fermenter (Biostat, Braun, Melsungen, Germany). For this purpose
100 g of dextrose and 11 L of water were sterilized in the
fermenter, and then a nitrogen assimilation medium [14.3 g yeast
nitrogen base (Life Technologies, Paisley, United Kingdom), 10 mg of
L-histidine, 20 mg of tryptophane, 20 mg of methionine, and 9 g of 15N-ammonia sulfate (95 atom%, Medgenix)] dissolved
in 400 mL of water was injected and inoculated with yeast. After 2 d at 25°C, when growth had reached a plateau, yeast was harvested,
sterilized, and lyophilized. One batch yielded about 50 g of
15N-enriched yeast. 15N-labeled sow's milk was
then produced by feeding two sows meals containing 20 g of
15N-labeled dry yeast and 2 L of 15N-labeled
cow's milk daily, 14 d long. Sows were separated from their
piglets and milked 12 h later. Milk flow was stimulated by
subcutaneous injection of 5 IU oxytocin (Alvetra, Neumünster,
Germany). The label of the pooled sow's milk was 0.697 atom%.
15N-casein was isolated from 15N-labeled cow's
milk by isoelectric precipitation at pH 4.6 (Michaelis and Pechstein 1912
), and 15N-lactoferrin from cow's or
sow's milk by ion exchange chromatography (Okonogi et al. 1988
) and proteins were lyophilized. All chemicals were of
analytical grade (Merck, Darmstadt, Germany).
Animals
Two separate experiments were done, one utilizing 43 suckling miniature pigs (age 10 d, both sexes), the other using nine adult male miniature pigs (age 15 mo) fitted with a T-canula at the terminal ileum. The animals were bred in our animal facility from a strain supplied by the Institut für Tierzucht und Haustiergenetik, Universität Göttingen, Germany. Both the adult animals and the piglets with their mothers were individually housed in different rooms maintained at 21°C and 55–70% relative humidity.
All experimental procedures described followed the established guidelines for the care and use of laboratory animals and were approved by the Animal Care and Animal Ethics Committee of the Ministry of Environment of Schleswig-Holstein, Germany.
Experimental Procedure and Diets
Suckling miniature pigs. 120 mg of 15N-labeled protein (porcine or bovine lactoferrin or bovine casein) and 30 mg chromic oxide were added to 10 mL of unlabeled porcine milk. This milk was given to 12-h food-deprived sucklings via an esophageal tube. Later (150 min) piglets were anesthetized with Stresnil® and Hypnodil® (Janssen, Neuss, Germany). The small intestine was excised and divided into three parts of equal length. The chyme was removed from the sections, which were then rinsed with distilled water to recover residual material. Contents were immediately frozen in liquid nitrogen and stored at -20°C until lyophilization, ground and passed through a sieve (0.5-mm pore size) before analysis.
Adult miniature pigs.
Animals consumed a basal semisynthetic diet described in Table 1.
Water was consumed ad libitum. No meal was given in the evening before
the experiment. The two experimental morning meals (204 g) were applied
in a cross-over design, 2 wk apart. One contained 7.5% (g/100 g)
15N-labeled casein, the other 1.25%
15N-labeled bovine lactoferrin. Both were adjusted to 15%
total protein with nonlabeled casein. Ileal chyme was collected over
the following postprandial 33 h, by blocking the flow of chyme
distal to the fistula with a balloon catheter (Size 14; Rüsch,
Kernen, Germany) (Schmitz et al. 1991
). As previous
experiments had shown that this block is incomplete, chromic oxide (20
g/kg) was added to the diet as an indigestible marker to correct for
losses. Chyme appearing at the fistula was immediately frozen and
stored at -20°C until lyophilization. The freeze-dried samples
of the first 3 h and the following six 5-h periods were pooled.
Freeze-dried chyme was treated as described before.
|
Total nitrogen of the diet and freeze-dried chyme samples
was determined using the Kjeldahl method. Chromic oxide was measured
according to Clarkson (1967)
. Measurement of
15N/14N isotope ratio and calculation of
protein digestibility was done as described previously (Roos et al. 1994
).
Electrophoretic Analysis and Immunoblotting
Protein content of chyme was determined (Lowry et al. 1951
), and equal protein loadings (30 µg) were developed on
10% of acrylamide gels (Laemmli 1970
) and blotted onto
nitrocellulose. Production of an antiserum against purified bovine
lactoferrin (gift of Prof. B. Senft, Gießen, Germany) in rabbits and
immunoblotting procedures were essentially as described (Seyfert
et al. 1986
). An alkaline-phosphatase-coupled secondary
antibody was used to develop the blots either conventionally with
5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium chloride
(Sigma, München, Germany) as substrates, or with the luminescense
emitting ECF (Amersham Buchler, Braunschweig, Germany) according to the
manufacturer. In the latter case, signals were visualized and
quantified with the STORM-phosphoimager (Molecular Dynamics,
Krefeld, Germany). On model blots, the intensity of the
lactoferrin-specific signal increased linearily (r
= 0.98) with lactoferrin loadings from 0.1 to 1 µg per slot.
Chyme of piglets which had been given only sow's milk was used as
control. Virtually no immunoreactive response was detected in controls,
proving that no endogenous lactoferrin appeared in the intestine.
Calculations and Statistics
The calculation of protein digestibility is based on the
measured values of 15N enrichment and concentration of
chromic oxide in the diet and the chyme. It is assumed that the flow of
dietary protein and the indigestible marker chromic oxide in the
intestinal tract are comparable. This assumption is supported by
previous observations that protein digestibility in adult miniature
pigs measured by this method was not significantly different 3, 6 and
12 h postprandially (Roos et al. 1994
). Presumably
this applies to suckling animals as well. In suckling animals the
calculations of digestibility were done by comparing the ratio
15N/Cr2O3 in chyme with the ratio
15N/Cr2O3 in the diet. In adult
animals the calculation was based on absolute recovery of
15N and Cr2O3, simply to emphasize
the fact that not all the chyme was collected, but part of it did flow
past the fistula into the large intestine.
Data shown are means ± SEM. Statistical evaluation
was performed by analysis of variance (ANOVA) using the Statgraphics
statistical package version 6.1, 1993 (Statistical Graphics
Corporation, Rockwell, MD). Comparison of different dietary regimens in
adult miniature pigs was done by one-way ANOVA, followed by the
Scheffé range test (Scheffé 1953
). In
suckling miniature pigs, comparison of different intestinal parts and
dietary regimens was done by multiple analysis of variance followed by
the Scheffé test. Differences were considered significant at
P < 0.05.
|
Results |
|---|
|
TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Digestibility, i.e., the oro-ileal disappearance of the
proteins, was calculated as the difference between the
15N-intake in the meal and the total 15N
recovered up to the respective section with both values expressed
relative to the indigestible marker chromic oxide. Disappearance of
casein up to the medial small intestine was 87.2%, but that of porcine
and bovine lactoferrin was less than 50%. In the distal as compared to
the medial section, casein digestibility was higher by 10.6 ± 3.7%, while digestibility of porcine and bovine lactoferrin was higher
by 39.9 ± 6.9 and 32.5 ± 4.2%, respectively. Nevertheless,
digestibility of both porcine and bovine lactoferrin was significantly
lower than that of bovine casein in both sections (Table 2
).Virtually no chyme was found in the proximal part of the small
intestine at the time of sacrifice of the animals (data not given).
Figure 1
shows the immunoblot of pure bovine lactoferrin (lane Lf) and chyme
samples of the distal intestinal section of four animals (lanes 1 to
4). Both in the range of 84 kDa (molecular weight of lactoferrin) and
~40 kDa, antilactoferrin reactivity was found. According to this
technique 1.1% of the administered lactoferrin was undigested, whereas
3.4% was partly digested (Table 3
).
|
|
|
Chromic oxide recovery after 33 h of sampling indicates that
only 54.3 ± 8.8 and 50.2 ± 9.5% of the chyme could be
recovered after lactoferrin or casein feeding, respectively
(Table 4
),meaning that a large part of the digesta did indeed flow past the
T-canula. Corrected for chromic oxide recovery, 2.3 ± 0.4 and
14.3 ± 2.5 µmol 15N-excess remained in the
intestine after feeding lactoferrin or casein, respectively. This means
that 9.3 ± 1.9 and 6.1 ± 1.0% of 15N were
recovered and, vice versa, 90.7 ± 1.9% of lactoferrin and 93.9
± 1.0% of casein were digested (P > 0.05).
|
|
Discussion |
|---|
|
TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionREFERENCES |
|---|
), where the slaughter approach yielded digestibilities
of 94.1, 92.2 and 94.1%, depending on the time point of postprandial
sampling (3, 6 and 12 h, respectively). The previous paper
highlights at the same time the fact that, within a certain time frame,
the time point of sampling is not critical, as the ratio of chyme flow
out of the stomach to digestive processes in the small intestine is
fairly constant. This study clearly demonstrates that in 10-d-old suckling piglets the digestibility of lactoferrin is much lower than of the highly digestible milk protein casein, while there is no significant difference in adult miniature pigs.
A lower digestibility of lactoferrin is in line with results from
previous in vitro and in vivo studies. Britton and Koldovsky (1987a
, b)
tested lactoferrin digestibility in vitro in gastric, jejunal and ileal
flushes of rats. They found a low degradation of lactoferrin in
suckling but a high degradation in weanling rats. But even in weanling
animals, lactoferrin proteolysis was still lower than that of casein.
In a previous investigation by this group, bovine lactoferrin was also
recovered from the intestine of suckling 3-wk-old miniature pigs at
3 h postprandially. Some of the recovered material was intact
protein, some was hydrolyzed, but still biologically active, as
demonstrated by rocket immunoelectrophoresis (Schmitz et al. 1988
). In another study, in vitro tryptic digestion of
lactoferrin over 3 h yielded up to five different fragments. The
two largest fragments were relatively resistant toward further
proteolysis over 24 h (Brock et al. 1976
).
Resistance of a 40 kDa lactoferrin hydrolysate against in vitro
digestion with pancreatin was described by Görtler et al. (1988)
.
In the present study, too, this 40 kDa lactoferrin fragment was
identified in the chyme of the distal small intestine of suckling
piglets. In total, 4.5% of the administered lactoferrin was recovered
by the immunological method. The 40 kDa peptide amounted to 76% of the
total immunologically identified lactoferrin activity (Table 2)
. One
might wonder why results based on the immunological assay differ from
those based on the 15N-label. But proteolysis products of
lactoferrin with proven bactericide activity, e.g., lactoferricin
(molecular mass: 3195; Dionysius and Milne 1997
), may
not respond to the applied antiserum at all. This would mean that the
immunological technique applied here probably underestimates recovery
and overestimates digestibility. Nevertheless, it proves beyond doubt
that undigested lactoferrin and immunoreactive peptide fragments
remained in the chyme of suckling piglets. The digestibility estimate
of the present study with 15N-labeled lactoferrin is
somewhat lower than homoarginine-based figures (Hagemeister et al. 1987
). This previous application of the homoarginine
technique in suckling animals suggested a prececal digestibility of
lactoferrin of 89.4 ± 2.3%. But the homoarginine technique may
overestimate digestibility if the label is not 100% evenly
distributed, because digestion of labeled sections may be faster than
digestion of nonlabeled sections of the molecule.
Overall, all these digestibility figures derived from different methods
confirm our assumption that lactoferrin is to some degree resistant to
digestion. In conclusion our results show that lactoferrin is less
well-digested in suckling piglets as compared to casein, whereas
this is not true in adult miniature pigs. The lower prececal
digestibility in suckling piglets as compared to adult animals may be
due to the undeveloped digestive capacity of the gastrointestinal tract
(Tarvid et al. 1994a
, b
). The incomplete development of the gastrointestinal
tract in the sucklings could also be the explanation for the
significantly lower digestibility of bovine casein up to the medial as
compared to the distal section, which was not found in adult miniature
pigs (Roos et al. 1994
). It might be assumed that
lactoferrin is more important for the newborn animal to protect it
against bacterial infections via the intestinal tract. The present
study finds no difference in the digestibility of lactoferrin of bovine
and porcine origin. From this result one may speculate that
lactoferrins of both species can exert bacteriostatic effects in the
intestine and that both homologous and heterologous lactoferrins may
have the potential to support the host defense system. This agrees with
findings of Teraguchi et al. (1993)
that bovine
lactoferrin did indeed effectively decrease the number of fecal
Enterobacteriaceae in mice.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Supported by the Deutsche Forschungsgemeinschaft
(Grant HA 456 2/2).
3 Purchase of the 15N-label was
supported by the H. Wilhelm Schaumann-Stiftung, Hamburg, Germany.
Manuscript received May 14, 1998. Initial review completed August 4, 1998. Revision accepted February 1, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMaterials and MethodsResultsDiscussionREFERENCES |
|---|
1. Abe H., Saito H., Miyakawa H., Tamura Y., Shimamura S., Nagao E., Tomita M. Heat stability of bovine lactoferrin at acidic pH. J. Dairy Sci. 1991;74:65-71[Abstract]
2.
Britton J. R., Koldovsky O. Luminal digestion of lactoferrin in suckling and weanling rats. Am. J. Physiol. 1987;253 (Gastrointest. Liver Physiol. 16):G397-G403
3. Britton J. R., Koldovsky O. Luminal digestion of human milk proteins in suckling and weanling rats. Nutr. Res. 1987;7:1041-1049
4. 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]
5. Clarkson E. M. A rapid method for the determination of chromium sesquioxide in faecal homogenates. Clin. Chim. Acta. 1967;16:186-188[Medline]
6.
Dionysius D. A., Milne J. M. Antibacterial peptides of bovine lactoferrin: purification and characterization. J. Dairy Sci. 1997;80:667-674
7. Ellison III R. T., Giehl T. J. Killing of gram-negative bacteria by lactoferrin and lysozyme. J. Clin. Invest. 1991;88:1080-1091
8. Görtler I., Bindels J. G., Sawatzki G. In vivo- und in vitro-Verdauung von Lactoferrin. Ernährungsumschau 1988;35:159(abs.)
9. Hagemeister H., Schmitz M., Schoppe I., Barth C. A. Absorption of oral bovine lactoferrin in the neonatal and adult pig. Proc. Nutr. Soc. 1987;46:110A
10. Hagiwara T., Shinoda I., Fukuwatari Y., Shimamura S. Effects of lactoferrin and its peptides on proliferation of rat intestinal epithelial cell line IEC 18, in the presence of epidermal growth factor. Biosci. Biotech. Biochem. 1995;59:1875-1881[Medline]
11. Hambraeus L. General discussion: Milk proteins and ligands. Barth C. A. Schlimme E. eds. Milk proteins 1988:110-111 Steinkopff & Springer Darmstadt, Germany, New York
12. Kinsella J. E., Whitehead D. M. Proteins in whey: chemical, physical, and functional properties. Adv. Food Nutr. Res. 1989;33:343-438[Medline]
13. Laemmli U. K. Cleavage of the structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970;227:680-685[Medline]
14.
Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1951;193:265-275
15. Manners M. J. The development of digestive function in the pig. Proc. Nutr. Soc. 1976;35:49-55[Medline]
16. Masson P. L., Heremans J. F., Dive C. An iron-binding protein common to many external secretions. Clin. Chim. Acta 1966;14:735-739
17. Michaelis L., Pechstein H. Der isoelektrische Punkt des Caseins. Biochem. Z. 1912;47:260-268
18. Okonogi, S., Tomita, M., Tomimura, T., Tamura, Y. & Mizota, T. (1988) A process for producing bovine lactoferrin in high purity. EPA 0 253 395 A1.
19. Roos N., Mahé S., Benamouzig R., Sick H., Rautureau J., Tomé D. 15N-labeled immunoglobulins from bovine colostrum are partially resistant to digestion in human intestine. J. Nutr. 1995;125:1238-1244
20. Roos N., Pfeuffer M., Hagemeister H. Labeling with 15N as compared with homoarginine suggests a lower prececal digestibility of casein. J. Nutr. 1994;124:2404-2409
21. Scheffé H. A method for judging all contrasts in the analysis of variance. Biometrica 1953;40:87-104
22. Schmitz M., Hagemeister H., Erbersdobler H. F. Homoarginine labeling is suitable for determination of protein absorption in miniature pigs. J. Nutr. 1991;121:1575-1580
23. Schmitz M., Hagemeister H., Görtler I., Bindels J. G., Barth C. A. Does bovine lactoferrin resist absorption in the small intestine of neonatal and adult pigs?. Barth C. A. Schlimme E. eds. Milk proteins 1988:103-104 Steinkopff & Springer Darmstadt, Germany, New York
24. Seyfert H. M., Sawatzki G. An estimation of the soluble tubulin content in Tetrahymena cells of normal and of size altered phenotype. Exp. Cell Res. 1986;162:86-96[Medline]
25. Tarvid, I., Cranwell, P. D., Ma, L. & Vavala, R. (1994) The early postnatal development of protein digestion in pigs. I. Pancreatic enzymes. Proceedings of the Sixth International Symposium on Digestive Physiology in Pigs, EAAP publication No. 80: 199–202.
26. Tarvid, I., Cranwell, P. D., Ma, L. & Vavala, R. (1994) The early postnatal development of protein digestion in pigs. II. Small intestinal enzymes. Proceedings of the Sixth International Symposium on Digestive Physiology in Pigs, EAAP publication No. 80: 181–184.
27. Teraguchi S., Ozawa K., Yasuda S., Fukuwatari Y., Shimamura S. Effects of orally administered bovine lactoferrin on the faecal Enterobacteriaceae of SPF mice fed milk. Biosci. Biotech. Biochem. 1993;57:360-361
28. Tomita M., Bellamy W., Takase M., Yamauchi K., Wakabayashi H., Kawase K. Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. J. Dairy Sci. 1991;74:4137-4142[Abstract]
This article has been cited by other articles:
|
|
 |
|
  H. Kuwata, K. Yamauchi, S. Teraguchi, Y. Ushida, Y. Shimokawa, T. Toida, and H. Hayasawa Functional Fragments of Ingested Lactoferrin Are Resistant to Proteolytic Degradation in the Gastrointestinal Tract of Adult Rats J. Nutr., August 1, 2001; 131(8): 2121 - 2127. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
