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Experimental Physiology 91.6 pp 1033-1040
DOI: 10.1113/expphysiol.2006.034876
© The Physiological Society 2006
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Enteric-formulated lactoferrin was more effectively transported into blood circulation from gastrointestinal tract in adult rats

 Takashi Takeuchi1, Takahiro Jyonotsuka1, Nao Kamemori1, Genji Kawano2, Hirohiko Shimizu2, Kunio Ando2 and Etsumori Harada3

Department of 1 Veterinary Medicine, Tottori University, Tottori 680-8553, Japan 2 NRL Pharma Inc., Kawasaki 213-0012, Japan 3 Rakuno-Gakuen University, Ebetsu 069-8501, Japan


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We have previously demonstrated that intestinally infused bovine lactoferrin (bLF) is transported into the blood circulation via the lymphatic pathway, not via the portal circulation. Therefore, in the present study, we further investigated whether intragastrically infused enteric-formulated bLF (EF-bLF) was more efficiently absorbed than bLF from the intestine in adult rats. The rats were randomly divided into three groups: 30 and 300 mg kg–1 non-enteric-formulated bLF (non-EF-bLF) groups and a 30 mg kg–1 EF-bLF group. Thoracic lymph was collected from a thoracic lymph duct under general anaesthesia. Bovine lactoferrin was infused into the stomach or duodenal lumen via a needle for a period of over 1 min in a volume of 1 ml kg–1. The bLF transported into the lymph was assayed quantitatively by double-antibody enzyme-linked immunosorbent assay (ELISA). Following the intragastric administration of bLF, the three groups showed almost the same lymph flow, but the bLF concentration in the lymph fluid in the EF-bLF group increased significantly and peaked 3 h after administration. With intraduodenal administration, the bLF concentration in the lymph fluid of the higher non-EF-bLF group was significantly higher than those of the other groups. The amount of absorbed bLF in the EF-bLF group was, however, about 10 times higher than that in the lower non-EF-bLF group, when it was administered intragastrically. These data show that enteric-formulated bLF is less susceptible to gastric pepsin and is more efficiently absorbed from the intestine than is non-enteric-formulated bLF.

(Received 24 June 2006; accepted after revision 1 September 2006; first published online 7 September 2006)
Corresponding author T. Takeuchi: Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan. Email: takeuchi{at}muses.tottori-u.ac.jp.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Oral administration of protein delivery have several limitations. These include low oral bioavailability of protein owing to degradation in the stomach, inactivation and digestion by proteolytic enzymes in the intestinal lumen, and poor permeability across the intestinal epithelium because of the high molecular weight and lack of lipophilicity (Paulsen et al. 1979; Maberly et al. 1982; Saffran et al. 1986). Therefore, a more efficient method, that is, a more effective protein delivery system, has been the subject of study. Many attempts have been made to deliver lactoferrin (LF) orally. One of them used enteric-formulated (EF) capsules, protected in liposomes (Ishikado et al. 2005); in this study, orally administered liposomal lactoferrin expressed more suppressive effects than non-liposomal lactoferrin on CCl4-induced hepatic injury in rats.

Recently, we reported that intraduodenally administered bovine LF (bLF) was successfully absorbed from the intestine via the lymphatic pathway in adult rats (Takeuchi et al. 2004). Moreover, Harada et al. (1999a) demonstrated that the LF contained in milk is transported into the circulation from the intestinal lumen and excreted into bile, suggesting the possibility of enterohepatic circulation of LF in neonatal pigs. In addition, orally administered LF was reported not only to be absorbed into the systemic circulation in neonates (Harada et al. 1999b; Talukder et al. 2002) but also to be absorbed in weaned pigs (Harada et al. 1999a) and young cattle (Talukder et al. 2003b). Talukder et al. (2003a) reported the detection of a specific LF receptor in the intestines, including the duodenum, jejunum, ileum and colon, and in Peyer's patches in the jejunum and ileum in the adult cow. Interestingly, the density of the LF receptor was higher in Peyer's patches than in other regions of the intestine. Thus, it is speculated that the LF transport mechanism is closely related to lymphatic organs in the weaned animal.

It is well known that orally administered LF has many effects. Moreover, much evidence has shown that a fragment of LF, named lactoferricin, is generated from digested LF and also has bioactive effects. Kuwata et al. (2001) demonstrated that lactoferrin and the LF fragment (lactoferricin) survived proteolytic degradation in the small intestine of adult rats. Hayashida et al. (2003a) determined the novel functions of orally administered bLF, such as an antinociceptive effect within the central nervous system of rats. This antinociceptive effect was induced not only by intraperitoneal injection (100 mg kg–1) but also by oral administration at doses of 1 g kg–1 (Hayashida et al. 2003a,b) or 300 mg kg–1 (Tsuchiya et al. 2006). However, these doses of bLF are too high for application in clinical treatment. Ishikado et al. (2005) reported the antigenicity of bLF. Their report indicated that orally administered bLF had less antigenicity than did subcutaneously administered bLF, and bLF had less antigenicity than did ovalbumin. Thus, formulations suitable for oral administration to ensure more efficient absorption of lactoferrin are essential, instead of the non-oral administration of lactoferrin at high doses.

The present study investigated whether intragastrically or intraduodenally administered bLF is absorbed into the lymph flow, while the dose dependency of the amount of an absorbed bLF and the efficiency of absorption of an enteric-formulated bLF (EF-bLF) were also studied. Moreover, we investigated the possibility of bLF storage in lymphocytes, to study its biological effects by comparing the number of lymphocytes in lymph fluid containing bLF with that of bovine serum albumin (BSA), and bLF contents within the lymphocytes collected from thoracic lymph following the administration of non-enteric-formulated bLF (non-EF-bLF) or EF-bLF.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Materials

Bovine lactoferrin was obtained from Tatua Biologics (Tatua, New Zealand). The purity of the bLF was 90% with an iron saturation of 15%. Coliforms, E. coli, S. aureus, Salmonella and Listeria were not detected, and yeasts and moulds were less than 1 cell g–1. Other chemicals were purchased from the following sources: Tris hydroxymethyl aminomethane was purchased from Wako Pure Chemical Co., Ltd (Osaka, Japan); bovine serum albumin (BSA) was purchased from Sigma (St Louis, MO, USA); mouse monoclonal antibody to bovine LF C-lobe was purchased from HyCult Biotechnology (Praha, Germany); and the enzyme-linked immunosorbent assay (ELISA) kit for bovine LF was purchased from Bethyl Laboratories, Inc.

Preparation of enteric-coated bLF

Enteric-coated bLF was obtained from Tatua Biologics (Tatua, New Zealand). Briefly, bLF powder was placed in soybean milk powder until EF-bLF contained 10% bLF. Details of the preparation of EF-bLF are described in international patent submission no. PCT/JP2006/301619. The diameter size of the EF-bLF was less than 1 mm, allowing it to pass through a size no. 16 mesh (pore diameter 1 mm).

Animals

Wistar strain male rats (220–280 g) were obtained from the Institute of Animal Reproduction (Ibaragi, Japan). The animals were maintained at a controlled temperature of 22 ± 2°C with a 12 h–12 h light–dark cycle (light cycle, 07.00–19.00 h), and were given standard chow (CE-2, Nihon Clea, Tokyo, Japan). The use of these animals, as well as the procedures performed, were approved by the Animal Research Committee at Tottori University.

Lymphatic bLF transport

Surgery.  All rats were fasted overnight, and then randomly divided into three groups: a 30 mg kg–1 non-EF-bLF (lower dose) group, a 300 mg kg–1 non-EF-bLF (higher dose) group and a 30 mg kg–1 EF-bLF group. Under 25% urethane (4 g kg–1, S.C.) and sodium pentobarbitone (Nembutal, 50 mg kg–1, S.C., Abbot Lab., North Chicago, IL, USA) general anaesthesia, a cannula was inserted into the external jugular vein in all rats to control the depth of anaesthesia by additional injection of pentobarbitone. The thoracic lymph duct was also cannulated with clear vinyl tubing (0.8 mm o.d.) in all rats according to the method of Bollman et al. (1948).

Experimental protocols.  The non-EF-bLF and the EF-bLF suspension were prepared in saline (0.9% NaCl). Under general anaesthesia, each solution or suspension was infused into the stomach or duodenum via a 26 gauge needle over a period of at least 1 min at a dose of 30 or 300 mg kg–1 body weight. Thoracic lymph was collected into heparin-coated tubes. Thoracic lymph fluid was harvested throughout the sampling period (1 h before and hourly after the bLF infusion for 4 h). The sampling tube was exchanged for a new one hourly. The flow rate of thoracic duct lymph was estimated by subtracting the weight of the empty tube from the total weight of the used tube. These samples were centrifuged at 5000 g at 4°C for 15 min. The supernatants and the lymphocyte pellets were stored at –80°C until analysis. The amounts of absorbed bLF in the thoracic duct lymph were calculated based on lymph flow rate and the concentration of bLF in the lymph.

Preparation of membrane and cytosolic fraction from thoracic lymphocytes.  For measurement of LF contents in the lymphocytes, thoracic lymph was collected following intraduodenal infusion of non-EF-bLF (30 mg kg–1) or BSA (30 mg kg–1, as a control). Following centrifugation of thoracic lymph fluid as described in the preceeding section, 1 ml of saline was added to the lymphocyte pellet in order to wash out the remaining bLF from the lymph fluid. After centrifugation for 10 min, the supernatant was discarded. The lymphocyte was resuspended in the 0.3 ml of fresh saline, then sonicated and centrifuged (12 000g for 10 min at 4°C). The supernatant was separated as a cytosolic fraction. The pellet was treated with 0.2% Triton X-100 solution (0.3 ml) and analysed as a membrane fraction.

ELISA for lactoferrin

Bovine lactoferrin absorbed into lymph was assayed quantitatively using a monoclonal antibody ELISA system. The minimum detectable quantity of bLF was 2 ng ml–1. The primary and secondary anti-bLF antibodies used in this ELISA system did not react with transferrin.

Statistical analysis

All data are expressed as means ± S.E.M. Differences between groups were assessed by repeated measured one-way ANOVA and Student's unpaired t test. A probability level of P < 0.05 was taken to be statistically significant in the analyses.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The lymph flow peaked 1 h after bLF administration in each group, and then gradually declined up to 4 h. There were no differences among the three groups regarding both the intragastric (F2,75 = 0.4980; P = 0.6254; Fig. 1A) and intraduodenal infusion (F2,75 = 0.2996; P = 0.7490; Fig. 1B).


Figure 1
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  		Figure 1.  Changes in the time course of the lymph flow of thoracic lymph fluid following intragastric (A) or intraduodenl infusion (B) of bLF
Lymph flow is expressed as total volume collected during each hour, and calculated per 100 g body weight. The figure shows results for the 30 ({circ}) and 300 mg kg–1 ({square}) non-EF-bLF groups and the 30 mg kg–1 EF-bLF group (•). Data represent the means ± S.E.M. from 5 rats.

 
Before the intragastric administration of bLF, no bLF was detected in all rats. The concentration of bLF in the thoracic duct lymph fluid in the lower dose non-EF-bLF group showed a very slight increase after administration. The higher dose non-EF-bLF group showed a clear increase in lymphatic bLF concentration during the first 1 h, and thereafter maintained the same level for 3 h. In contrast, a marked increase of bLF concentration was demonstrated in the EF-bLF group. A significant difference was found among the three groups by ANOVA (F2,75 = 37.02; P < 0.0001). The concentrations in the EF-bLF group were significantly high at 1–4 h after administration compared with the lower dose non-EF-bLF group. Although the bLF level for the first 1 h was similar to that in the higher dose non-EF-bLF group, a particularly high concentration of bLF was determined after 3 h (Fig. 2A).


Figure 2
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  		Figure 2.  Changes in the time course of bLF concentration in the thoracic lymph fluid following intragastric (A) or intraduodenal infusion (B) of bLF
Lymphatic bLF concentration is expressed as a mean during each hour. The figure shows results for the 30 ({circ}) and 300 mg kg–1 ({square}) non-EF-bLF groups and the 30 mg kg–1 EF-bLF group (•). Data represent the means ± S.E.M. from 5 rats. Significant differences from the 30 mg kg–1 non-EF-bLF group are shown as *P < 0.05 and **P < 0.01. Significant difference from the 300 mg kg–1 non-EF-bLF group is shown as ##P < 0.01.

 
In contrast, when bLF was administered intraduodenally, the lymphatic bLF concentration was dose dependent (F2,75 = 10.73; P = 0.0054; Fig. 2B). No bLF was detected before the administration in all groups. Moreover, the bLF concentration at 1 h in the EF-bLF group showed slightly diminished absorption compared with the other groups. Significantly higher bLF concentrations were seen in the higher dose non-EF-bLF group compared with the other groups for 1–3 h after administration.

The time course of the amount of bLF absorbed into the thoracic lymph was closely related to the concentration of bLF in the lymph fluid after administration (Fig. 3A). After intragastric administration, ANOVA showed a significant difference among the three groups (F2,75 = 17.40; P = 0.0012). The amounts of absorbed bLF in the higher dose non-EF-bLF group were significantly higher than those of lower dose non-EF-bLF group at 2 and 3 h. The amounts of absorbed bLF in the EF-bLF group were particularly high after 2 h compared with the lower dose non-EF-bLF group. In contrast, after intraduodenal administration, the amounts of absorbed bLF peaked at 1 h in all groups and then gradually declined up to 4 h (Fig. 3B). After duodenal administration, a significant difference among the three groups was found by ANOVA (F2,75 = 6.720; P = 0.0194). The amount of absorbed bLF in the higher dose non-EF-bLF group was significantly higher than those in the other groups at 1 h. The amount of absorbed bLF in the EF-bLF group showed no significant difference compared with that in the lower dose non-EF-bLF group.


Figure 3
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  		Figure 3.  Changes in the time course of bLF uptake into the thoracic lymph fluid following intragastric (A) or intraduodenal infusion (B) of bLF
The uptake of bLF into the lymph fluid is expressed as total volume during each hour, and calculated per 100 g body weight. The figure shows results for the 30 ({circ}) and 300 mg kg–1 ({square}) non-EF-bLF groups and the 30 mg kg–1 EF-bLF group (•). Data represent the means ± S.E.M. from 5 rats. Significant differences from the 30 mg kg–1 non-EF-bLF group are shown as *P < 0.05 and **P < 0.01. Significant difference from the 300 mg kg–1 non-EF-bLF group is shown as #P < 0.05.

 
The total amount of bLF absorbed into the thoracic lymph for the first 4 h following administration is shown in Fig. 4. The amount of absorbed bLF in the EF-bLF group was considerably higher than those in the other groups when it was administered intragastrically, being about 10 times higher than that in the lower dose non-EF-bLF group (F2,15 = 17.22; P = 0.0003; Fig. 4A). Intraduodenal administration also resulted in a significant difference among the three groups by ANOVA (F2,15 = 6.766; P = 0.0108). There were no significant differences, however, between the non-EF-bLF and EF-bLF at the same dose, when they were administered intraduodenally (Fig. 4B).


Figure 4
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  		Figure 4.  Total uptake of bLF into the thoracic lymph fluid for 4 h following intragastric (A) or intraduodenal infusion (B) of bLF
Total uptake of bLF is expressed as total volume over 4 h, and calculated per 100 g body weight. Data represent means ± S.E.M. from 5 rats. Significant difference from the 30 mg kg–1 non-EF-bLF group is shown as **P < 0.01. Significant difference from the 300 mg kg–1 non-EF-bLF group is shown as #P < 0.05.

 
As shown in Fig. 5, after the administration of non-EF-bLF (30 mg kg–1) or BSA (30 mg kg–1) into the duodenum, the number of lymphocytes in thoracic lymph fluid increased slightly. However, no significant difference was seen between the two groups. Most of the bLF was contained in the supernatant of the lymph fluid. Only 1–8% of the bLF was located in the lymphocytes after administration (Fig. 6B), and the ratio of bLF localization within the cell was similar in the membrane and cytosolic fractions (Fig. 6A).


Figure 5
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  		Figure 5.  Time course of the number of lymphocytes in the thoracic lymph following intraduodenal infusion of non-EF-bLF (•) or BSA ({circ}) at dose of 30 mg kg–1
Number of lymphocytes is expressed as the total number during each hour, and calculated per 1 µl. Data represent means ± S.E.M. from 5 rats.

 

Figure 6
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  		Figure 6.  Time course of bLF contents of lymphocytes in the thoracic lymph fluid following the intraduodenal infusion of bLF
The figure shows the localization of bLF contents within the supernatant of thoracic lymph fluid, the cell membrane and cytosolic fraction (A), and the percentage of bLF within the lymphocytes collected from the thoracic lymph fluid (B). The calculations of bLF contents and the percentage of bLF were performed using the total volume collected during each hour. Data represent means ± S.E.M. from 5 rats.

 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In a previous article, we reported that intra-duodenally administered bLF was transported into the blood circulation via the thoracic duct lymph fluid in adult rats (Takeuchi et al. 2004). Moreover, in that report, the plasma bLF concentration in the collected thoracic lymph group, in which the lymph fluid was sampled from the thoracic duct, did not increase after the intraduodenal administration of bLF, while it increased rapidly in the non-collected thoracic lymph group, in which the lymph fluid was not sampled. These findings indicate that bLF is transported to epithelial cells from the lumen and reaches the blood circulation via the lymphatic pathway, not via portal circulation. Thus, absorbed LF is not immediately metabolized in the liver, suggesting that, at an effective dose, it may be distributed to the whole body.

Wakabayashi et al. (2004) reported that a dietary LF or its fragments was not transferred to portal blood in healthy adult rats. In a preliminary study, we also tried to detect intestinally transferred LF or its fragments in portal blood in adult rats; however, we couldn't detect any LF particles using the ELISA system (data not shown). There are some reasons why intestinally administered LF has not been detected in the portal blood. First, the main route of intestinal transportation of LF is the lymphatic pathway (Takeuchi et al. 2004). Second, the transferred LF is rapidly cleared by hepatic uptake, having a half-life of 10 min in adult rats (Regoeczi et al. 1985).

In the present study, doses of 30 and 300 mg kg–1 of non-EF-bLF were used, and the bLF concentration of the lymph fluid in the higher dose non-EF-bLF group was higher than that in the lower dose non-EF-bLF group, not only when they were administered duodenally but also gastrically. The total amount of bLF absorbed into thoracic lymph was also similar. These results mean that a higher concentration of bLF within the small intestine induced a higher concentration of lymphatic bLF in the higher dose non-EF-bLF group. Kawakami & Lonnerdal (1991) have isolated the LF receptor in human fetal and infant small intestines. We have reported that intraduodenally administered bLF was absorbed via a receptor-mediated transporting system in rats (Takeuchi et al. 2004), pigs (Harada et al. 1999a) and cattle (Talukder et al. 2003b). Therefore, a higher concentration of bLF may have an advantage in absorbing from the small intestine. The concentrations and amounts of lymphatic bLF in the EF-bLF group showed no significant difference compared with that of the lower dose non-EF-bLF group when they were administered intraduodenally. In the small intestine, the concentration of bLF in the rumen is one of the main factors affecting the absorption activity.

In contrast, the amount of transferred bLF in the lymph fluid in this study was about four times higher than that in the previous study (Takeuchi et al. 2004). The lymph flow was also slightly higher in the present study than that in the previous report. One of the reasons for the discrepancies may be a difference of anaesthetic procedure: intraperitoneal injection of pentobarbitone in the previous study, compared with subcutaneous injection of urethane and pentobarbitone or intravenous injection of pentobarbitone in the present study.

The concentrations and the amounts of lymphatic bLF after gastric administration in the EF-bLF group were significantly higher than those of the higher dose non-EF-bLF group. These results clearly demonstrate that the EF-bLF had been protected from gastric digestion, resulting in greater absorption because of the higher concentrations in the small intestine. Generally, it is well known that enteric-formulated substances have a resistance to gastric acid and pepsin digestion. Kovacs-Nolan & Mine (2005) have reported that microencapsulation for the gastric passage and controlled intestinal release of immunoglobulin Y had resistance in the gastric condition for up to 6 h in vitro. We clearly demonstrated that the EF-bLF particle used in the present study was quite effectively absorbed from intestine.

The present study indicated that about 8% of absorbed bLF was located within the lymphocytes after intraduodenal administration (Fig. 6). The neutrophil is believed to be the largest storage for endogenous LF, although we cannot explain the precise physiological role of bLF-positive lymphocytes. However, the internalized bLF may have a longer half-life within the blood circulation, since free bLF will quickly be trapped in the liver (Regoeczi et al. 1985) and then excreted into the gall bladder. Prieels et al. (1978) demonstrated that about 90% of intravenously injected LF was cleared from the circulation by the liver in less than 10 min after administration, and Ziere et al. (1992) also showed that 92.8 ± 9.5% of intravenously injected LF was cleared from the circulation by the liver at 5 min after injection. This survival time is too short to affect each organ for a long period, so it can be expected that LF located within lymphocytes may have a long life and affect the organs after the clearance of free LF from the circulation.

Lactoferrin is well known as a multifunctional protein, having antibacterial, anti-inflammatory, iron-binding, antitumour, antinociceptive and antistress activities. Inoue et al. (2004) reported that the LF level in the blood increased after running exercise in humans. Some questions arise, including why endogenous LF is quickly released into the bloodstream, and why LF has a different function in each organ. These questions have yet to be clarified, but it may be necessary to release the endogenous LF, when the body requires a higher concentration of lactoferrin in the bloodstream. In a previous study, we demonstrated in rats that intraduodenally administered bLF was transported into thoracic lymph fluid, whence it might reach the whole body (Takeuchi et al. 2004). Although the exogenously applied dietary LF was very small amount, it induced several pharmacological functions. Further experiments are required to clarify this point.

Several phenomena may provide support for the role of lymphocytes regarding these issues. Iigo and co-workers have reported that LF has cancer-preventive effects on the production of cytokines (Iigo et al. 1999, 2004). Takeuchi et al. (2003) have reported that bLF suppressed distress induced by maternal separation via an opioid-mediated mechanism. They also indicated that bLF possibly activates nitric oxide synthase, and elevated nitric oxide may lead to some modification of the opioid system. Lactoferrin may affect some organs via the production of various cytokines, which have many biological effects. Further investigation is necessary to clarify the precise role of lymphocytes in modifying the LF transport in the blood.

In conclusion, the present study demonstrates that a higher dose of bLF results in a higher concentration of lymphatic bLF in adult rats, while EF-bLF resists gastric digestion and is efficiently absorbed, whereas non-EF-bLF is susceptible to degradation by gastric digestion. Thus, EF-bLF is more effectively transported into the blood circulation from the gastrointestinal tract than non-EF-bLF.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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   Acknowledgements
 
Part of this investigation was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 17580256).





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expphysiol.2006.034876v1
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