Vol. 282, Issue 4, G711-G719, April 2002
Electrical charge on protein regulates its absorption from
the rat small intestine
Makiya
Nishikawa1,
Susumu
Hasegawa1,
Fumiyoshi
Yamashita1,
Yoshinobu
Takakura2, and
Mitsuru
Hashida1
Departments of 1 Drug Delivery Research and
2 Biopharmaceutics, Graduate School of
Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
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ABSTRACT |
The effect of the electrical charge on the
intestinal absorption of a protein was studied in normal adult rats.
Chicken egg lysozyme (Lyz), a basic protein with a molecular weight of
14,300, was selected and several techniques for chemical modification were applied. Then the intestinal absorption of Lyz derivatives was
evaluated by measuring the radioactivity in plasma and tissues, after
the administration of an 111In-labeled derivative to an in
situ closed loop of the jejunum. After the administration of
111In-Lyz, the level of radioactivity in plasma was
comparable with the lytic activity of Lyz, supporting the fact that the
radioactivity represents intact Lyz. 111In-cationized Lyz
showed a 2-3 times higher level of radioactivity in plasma,
whereas the radioactivity of 111In-anionized Lyz was much
lower. The absorption rate of 111In-Lyz derivatives
calculated by a deconvolution method was correlated for the strength of
their positive net charge. A similar relationship was observed using
superoxide dismutase. These findings indicate that the intestinal
absorption of a protein is, at least partially, determined by its
electrical charge.
intestinal absorption; chicken egg lysozyme; pharmacokinetics; chemical modification; superoxide dismutase
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INTRODUCTION |
INTESTINAL PERMEABILITY
IS known to be altered in disease states such as Crohn's
disease, celiac disease, viral infection, and multiple organ failure
(38, 42, 47). Under these abnormal conditions, not only
low-molecular weight compounds but also macromolecules, such as
proteins, can pass transcellularly or paracellularly through the
intestinal epithelium and be absorbed into the circulation. Altered
properties of the intestinal epithelial cells would explain the
difference in permeability. Such changes lead to the increased nonspecific adsorption of proteins to the cell (42).
Increased permeability of macromolecules is related to the symptoms
observed in these diseases.
However, in healthy adults, it is well known that the intestinal
permeability of a solute through the intercellular junction (paracellular pathway) is highly dependent on its molecular size (6, 15). Molecules that hardly interact with intestinal
tissue might exhibit a simple correlation between their molecular size and rate and extent of absorption. Therefore, the intestinal epithelium of healthy adults is generally considered to be virtually impermeable to macromolecules. In addition to this mechanical barrier presented by
the tissue, the enzymatic barrier, i.e., a rapid and extensive degradation of proteins by digestive enzymes, highly restricts the
entry of intact, undigested proteins into the body (42). However, some studies support the idea that there is a small, but
significant, degree of transport of biologically and/or antigenically active peptides and proteins through the epithelial cells of the intestines (2, 5, 17, 25, 41). The mechanism of this transport through the intestines has received little attention to date.
Intestinal epithelial cells possess a negatively charged cell surface
as do other types of cells. The charged surface provides sites of
interaction for positively charged compounds. Because of this
charge-based interaction, cationic macromolecules have been used to
increase the delivery of drugs and genes to target cells (3,
4). Positive charges on the molecular surface can be
electrostatically attracted and adsorbed to the negatively charged cell
surface glycoproteins, followed by increased cellular uptake of the
positively charged molecules. These findings suggest that electrostatic
interaction of protein with the intestinal epithelial cells may be a
factor determining its intestinal absorption. However, there are few
investigations of the intestinal absorption of proteins that have
considered the electrical charge. Proteins formulated in oral dosage
forms include positively charged ones, such as lysozyme, bromelain, and
pancreatopeptidase E. The cationic nature of these protein drugs could
facilitate their interaction with intestinal tissue, resulting in
detectable absorption from the intestine (5). The effect
of the electrical charge of proteins on their intestinal absorption
needs to be quantitatively investigated.
To this end, we chose chicken egg lysozyme (Lyz) as a model positively
charged protein (isoelectric point of 11) with a molecular weight of
14,300, because it can be absorbed from the intestines in small
quantities (53, 54). Its electrical charge is altered by
chemical modification, i.e., coupling with hexamethylenediamine or
succinic anhydride to endow Lyz with an additional positive charge or
negative charge, respectively. In addition, galactose or glucose can be
covalently attached to Lyz to give glycosylated derivatives, because
the intestinal epithelial cells possess glucose transporters, and some
reports (23, 30) suggested their involvement in the
enhanced absorption of glycosylated molecules. Pharmacokinetic profiles
of these derivatives radiolabeled with 111In were studied
in rats after intrajejunal administration or intravenous injection. The
absorption rate was estimated by a deconvolution method using the
profiles of the concentrations in plasma after intravenous and
intrajejunal administration, and the relationship between the
electrical charge of the protein derivatives and their absorption rate
from the intestine was examined. In addition, to examine whether the
relationship obtained can be applied to other proteins, we also report
the altered intestinal absorption properties of recombinant human
superoxide dismutase (SOD) that is different from Lyz in its
physicochemical properties such as the electrical charge (negative,
isoelectric point of ~5) and molecular weight (32,000)
after cationization.
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MATERIALS AND METHODS |
Animals.
Male Wistar rats (180-210 g) were purchased from the Shizuoka
Agricultural Cooperative Association for Laboratory Animals (Shizuoka,
Japan). Rats were fasted for 20 h before experimentation. All
procedures were examined by the Ethics Committee on Animal Experimentation at Kyoto University.
Chemicals.
Chicken egg Lyz and FITC were purchased from Sigma (St. Louis, MO).
Recombinant human SOD (111Ser) was supplied by Asahi
Chemical (Shizuoka, Japan). Diethylenetriaminepentaacetic acid (DTPA)
anhydride was purchased from Dojindo Laboratory (Kumamoto, Japan).
111InCl3 was supplied by Nihon Medi-Physics
(Takarazuka, Japan). Micrococcus lysodeikticus was purchased
from Nacalai Tesque (Kyoto, Japan). All other chemicals were obtained
commercially as reagent-grade products.
Synthesis of Lyz and SOD derivatives.
Anionized Lyz (An-Lyz) was synthesized by succinylation
(51), i.e., by reacting succinic anhydride with the amino
group of Lyz. Coupling 1,6-hexamethylenediamine to Lyz was performed with 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide to obtain
highly positively charged Lyz [cationized Lyz (Cat-Lyz)]
(49). Glucosylated (Glc-Lyz) and galactosylated
Lyz (Gal-Lyz) were synthesized by reacting Lyz with
2-imino-2-methoxyethyl 1-thioglucoside or thiogalactoside, respectively, according to the method of Lee et al. (24).
Highly negatively charged SOD [anionized SOD (An-SOD)] and cationized SOD (Cat-SOD) were synthesized by the same method as An-Lyz and Cat-Lyz, respectively.
The number of amino groups in each derivative was determined by
trinitrobenzene sulfonic acid using glycine as a standard (11). The number of sugar residues was determined by the
anthron-sulfuric acid method. The molecular weight of Lyz derivatives
was estimated by SDS-PAGE using a standard curve obtained with marker
proteins (low-range marker; Wako, Osaka, Japan), and that of
SOD derivatives was estimated by HPLC gel-filtration chromatography
using Shim-pack Diol-300 column (Shimadzu, Kyoto, Japan).
Electrophoretic mobility of Lyz and SOD derivatives was measured with a
laser electrophoresis-zeta potential analyzer (LEZA-500T; Otsuka
Electronics). The lytic activity of Lyz derivatives was measured using
M. lysodeikticus according to the method of Selsted and
Martinez (45).
Labeling.
Lyz and SOD derivatives were radiolabeled with 111In using
the bifunctional chelating agent, DTPA anhydride, according to the method of Hnatowich et al. (16). In brief, protein (2 mg)
was dissolved in 1 ml 4-(2-hydroxyethyl)-1-piperazinethane sulfonic acid buffer (0.1 M, pH 7.0) and a twofold molar excess of DTPA anhydride in 10 µl dimethyl sulfoxide was added. After stirring for
30 min at room temperature, the mixture was purified by gel-filtration chromatography using a Sephadex G-25 column (1 × 40 cm) and
eluted with acetate buffer (0.1 M, pH 6.0) to separate unreacted DTPA. Fractions containing DTPA-coupled protein were selected using spectrophotometry and concentrated by ultrafiltration. Thirty microliters 111InCl3 solution was added to 30 µl sodium acetate buffer (1 M, pH 6.0), and 60 µl DTPA-protein
derivative was then added to the mixture. After 30 min at room
temperature, the mixture was purified by gel filtration chromatography
using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and
eluted with acetate buffer (0.1 M, pH 6.0). The appropriate fractions
were selected based on their radioactivity and concentrated by
ultrafiltration. The specific activity of the obtained samples was
~40 MBq/mg protein.
Separately, FITC was coupled to Lyz derivatives by the method of
Monsigny et al. (31) for confocal fluorescence microscopic studies.
Biodistribution after intravenous injection.
Rats were anaesthetized by intraperitoneal injection of pentobarbital
sodium (50 mg/kg). The urinary bladder and bile duct were cannulated
for the collection of bile and urine samples. 111In-Lyz
derivative was injected into the femoral vein at a dose of 0.1 mg/kg.
At predetermined time points, blood, urine, and bile were collected for
the entire experimental period (3 h). At the end, rats were killed, and
the liver, kidney, and spleen were sampled. Blood samples were
centrifuged at 2,000 g for 2 min, and 100 µl plasma was
assayed for radioactivity.
Intestinal absorption from an in situ closed loop.
Intestinal absorption of the test compound was examined in the in situ
closed loop of the jejunum. A midline abdominal incision was made, and
the lumen of the jejunum was washed with saline three times. A jejunal
loop, 5 cm in length, was prepared by closing both ends with sutures.
Each protein derivative was dissolved in 700 µl phosphate buffer
(0.15 M, pH 6.5) and then administered into the jejunal loop at a dose
of 1 or 10 mg/kg body wt. Blood, urine, and bile samples were collected
for 3 h.
Degradation of 111In-Lyz derivatives in the loop was
examined in different rats. At 30 min, 1 or 3 h after intrajejunal
administration, the contents of the loop were subjected to
gel-filtration chromatography on a Sephadex G-50 column (1 × 40 cm) and eluted with MES buffer (0.05 M, pH 6.0).
Determination of 111In radioactivity and lytic
activity of Lyz derivatives.
111In radioactivity in each sample was measured in a
well-type NaI scintillation counter (ARC-500, Aloka, Tokyo). Lytic
activity of Lyz and Cat-Lyz in plasma was measured using M. lysodeikticus as described above.
Confocal microscopic images.
FITC-Lyz derivatives were introduced into the loop in the same manner
as in the in situ absorption experiment. At 1 h after administration, rats were killed and the loop was excised, washed with
PBS, and frozen. Cryosections 10-µm thick were made using a cryostat
(CM3000-Kryostat; Leica, Heidelberg, Germany) and fixed with 4%
formaldehyde; the nucleus was stained with propidium iodide. Slices
were scanned with a confocal laser microscope (ACAS 570 interactive
laser cytometer; Meridian Instruments).
Estimation of amount absorbed by deconvolution method.
Plasma concentration of 111In-protein after intrajejunal
administration [Cpo(t)] is expressed as
follows (39)
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(1)
|
where f(t) is an absorption rate at time t
and Civ(t) is the plasma concentration at
time t after intravenous injection of an unit dose (an
impulse input). When the amount of f(
)
is rapidly injected to
the systemic circulation at time , plasma concentration
associated with the pulse is
f()Civ(t
) at time t.
Equation 1 is derived, considering that an absorption
rate-time profile is composed of an infinite number of the input
pulses. Absorption profiles of 111In-protein were estimated
by deconvoluting Cpo(t) with
Civ(t) in Eq. 1 (20). In
applying the computation algorithm (20), the plasma
concentration-time profile of radioactivity after intravenous injection
of 111In-protein was approximated with a biexponential equation.
Statistical analysis.
Differences were statistically evaluated by one-way ANOVA followed by
the Student-Newman-Keuls multiple comparison test. The level of
significance was set at * P < 0.05 and
** P < 0.01.
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RESULTS |
Physicochemical characteristics of Lyz derivatives.
The physicochemical characteristics of Lyz derivatives are summarized
in Table 1. All synthesized Lyz
derivatives had a similar molecular size to unmodified Lyz. The number
of free amino groups in Lyz fell from 7.8 to 1.4 for An-Lyz and to 2.5 for Glc- and Gal-Lyz, whereas the number increased to 10.5 for Cat-Lyz.
Lyz had an electrophoretic mobility of 0.14 × 10
4
cm2 · V1 · s1
at pH 6.5, after determination by a zeta potential analyzer. The
mobility increased by cationization to 0.56 × 104
cm2 · V1 · s1.
On the other hand, An-Lyz was electrophoresed to the positive pole,
indicating its negative surface charge (0.52 × 104
cm2 · V1 · s1
). These determinations confirmed that Cat-Lyz is a highly
cationic derivative of Lyz, whereas An-Lyz is an anionic
derivative. Glycosylation slightly reduced the positive charge of Lyz.
Lytic activity was almost unchanged for Cat-, Glc-, and Gal-Lyz.
However, succinylation of Lyz (An-Lyz) resulted in a complete loss of
enzymatic activity, suggesting the cationic charge of Lyz is critical
for its lytic activity.
Disposition of 111In-Lyz derivatives after intravenous
injection.
The tissue disposition of 111In-Lyz derivatives was
examined after intravenous bolus injection into rats. Figure
1A shows the plasma concentration-time
profile of radioactivity after intravenous injection at a dose of 0.1 mg/kg into rats. 111In-Lyz rapidly disappeared from plasma.
Chemical modification of Lyz slightly altered the elimination rate from
plasma and had a marked effect on the tissue disposition of
111In-Lyz (Fig. 1, B-F).
111In-Lyz was recovered largely in the kidneys (53% of
dose) and urine (20%) but not in the liver (1.9%), reflecting its
high susceptibility to glomerular filtration and reabsorption
(18, 26). 111In-An-Lyz was largely excreted
into urine instead of accumulating in the kidney, whereas
111In-Cat-Lyz was recovered in both the liver (31%) and
kidney (54%). 111In-Gal-Lyz and 111In-Glc-Lyz
also showed high hepatic recovery in addition to recovery in the kidney
and urine, suggesting their recognition by the asialoglycoprotein receptors on hepatocyte (34).
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Fig. 1.
Plasma concentration-time course (A) and tissue
disposition at 3 h (B-F) of radioactivity
after intravenous injection of 111In-Lyz derivatives into
rats at a dose of 0.1 mg/kg.  , B,
111In-Lyz;  , C,
111In-An-Lyz;  , D,
111In-Cat-Lyz;  , E,
111In-Glc-Lyz;  , F,
111In-Gal-Lyz. Results are expressed as means ± SD of
3 rats. * P < 0.05 and ** P < 0.01, statistically significant difference from 111In-Lyz.
The concentration of 111In-Lyz derivatives in plasma was
also significantly different from 111In-Lyz although not
marked in the figure: 111In-An-Lyz (P < 0.05 for 5 min, 3 h; P < 0.01 for 30 min, 1 and
2 h); 111In-Cat-Lyz (P < 0.05 for 30 min, 3 h; P < 0.01 for 1 and 2 h);
111In-Glc-Lyz (P < 0.05 for 1 and 3 h; P < 0.01 for 2 h); and
111In-Gal-Lyz (P < 0.05 for 10, 30 min,
and 3 h; P < 0.01 for 2 and 5 min, 1 and 2 h). Lyz, lysozyme; An, anionized; Cat, cationized; Glc, glucosylated;
Gal, galactosylated.
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Tissue disposition of 111In-Lyz after intrajejunal
administration.
111In-Lyz was administered into the jejunal loop, and the
contents of the loop were applied to a column to check the molecular size of 111In-Lyz. 111In-Lyz recovered at 0.5 and 1 h after its administration into the jejunal loop showed a
similar chromatographic profile to that of intact
111In-Lyz. At the end of the 3-h experiment, ~3% of the
radioactivity was eluted in fractions different from those of
111In-Lyz (data not shown).
Figure 2A shows the plasma
concentration-time profile of radioactivity after intrajejunal
administration of 111In-Lyz derivatives at a dose of 1 mg/kg. Radioactivity was detected in plasma after intrajejunal
administration of 111In-Lyz, indicating that
111In-Lyz can be absorbed from the intestine. Compared with
111In-Lyz, 111In-Cat-Lyz showed a two- to
threefold higher level of radioactivity in plasma (P < 0.05 at 2 h, P < 0.01 at 1 and 3 h). On the
other hand, 111In-An-Lyz showed much less radioactivity,
although the difference was not significant. The plasma level of
radioactivity after the administration of the
111In-glycosylated Lyz derivatives was a little lower than
that of 111In-Lyz. The tissue disposition profiles of
radioactivity after intrajejunal administration of the
111In-Lyz derivatives were similar to those obtained after
administration by the intravenous route (Fig. 2,
B-F). These tissue disposition data after
intrajejunal administration suggest that Lyz derivatives retain their
structural characteristics even after passage through the intestinal
epithelial cells into the blood circulation.
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Fig. 2.
Plasma concentration-time course (A) and tissue
disposition at 3 h (B-F) of radioactivity
after intrajejunal administration of 111In-Lyz derivatives
into rats at a dose of 1 mg/kg. , B,
111In-Lyz; , C,
111In-An-Lyz; , D,
111In-Cat-Lyz; , E,
111In-Glc-Lyz; , F,
111In-Gal-Lyz. Results are expressed as means ± SD of
3 (111In-An-Lyz), 4 (111In-Lyz,
111In-Cat-Lyz, 111In-Gal-Lyz), or 6 (111In-Glc-Lyz) rats. * P < 0.05 and
** P < 0.01, statistically significant difference
from 111In-Lyz.
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When the tissue disposition was normalized with respect to the dose
(%dose or %dose/ml), no significant differences were observed in the
tissue disposition of radioactivity after intrajejunal administration
of 111In-Lyz or 111In-Cat-Lyz at a dose of 1 and 10 mg/kg (Fig. 3). For both
derivatives, the disposition profiles at the 10 mg/kg dose could be
superimposed on those obtained at the 1 mg/kg dose, indicating that the
intestinal absorption of these Lyz derivatives is proportional to the
dose over the range examined.
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Fig. 3.
Dose dependence of plasma concentration-time course of
radioactivity after intrajejunal administration of
111In-Lyz (A) and 111In-Cat-Lyz
(B) into rats at a dose of 1 or 10 mg/kg. ,
1 mg/kg; , 10 mg/kg. Results are expressed as
means ± SD of 3 (10 mg/kg) or 4 (1 mg/kg) rats.
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To see whether the absorbed fraction of 111In-Lyz remains
intact, the lytic activity of Lyz in plasma after intrajejunal
administration of (nonradiolabeled) Lyz was examined at a dose of 10 mg/kg. The concentration of lytic activity in plasma was comparable
with, and not significantly different from, the concentration of
111In radioactivity after 111In-Lyz
administration, after their normalization to the administration dose
(Fig. 4). These results suggest that the
radioactivity in plasma after administration of 111In-Lyz
is due to intact Lyz.
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Fig. 4.
Comparison of radioactivity and lytic activity of Lyz
after intrajejunal administration of 111In-Lyz and Lyz,
respectively, into rats at a dose of 10 mg/kg. , lytic
activity; , 111In radioactivity. Results
are expressed as means ± SD of 4 (111In
radioactivity) or 3 (lytic activity) rats.
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Confocal microscopic images of rat jejunum after administration of
FITC-Lyz derivatives.
Figure 5 shows the confocal microscopic
images of rat jejunum cryosections after intrajejunal administration of
FITC-Cat-Lyz. Green fluorescence derived from FITC-Cat-Lyz was mainly
observed around the luminal surface of the tissue. In addition,
fluorescence could be detected near the nucleus of the epithelial
cells. Fluorescent intensity associated with the intestinal tissue
depended on the electrical charge of the Lyz derivative, and
FITC-An-Lyz had a much weaker signal (data not shown).
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Fig. 5.
Confocal microscopic images of cryosections of the rat jejunum
after intrajejunal administration of FITC-Cat-Lyz to rats. Nuclei of
cells were stained with propidium iodide. Left, ×169
maginification; right, ×564 magnification of rectangular
area indicated by the white line, left.
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Intestinal absorption rate of 111In-Lyz derivatives
calculated by deconvolution method.
Intestinal absorption-time courses of 111In-Lyz derivatives
were calculated by a deconvolution method using the plasma
concentration-time profiles after intravenous and intrajejunal
administration (Fig. 6).
111In-Lyz derivatives were linearly absorbed from the
jejunum with time. The amount of 111In-Cat-Lyz absorbed was
significantly greater than that of 111In-Lyz
(P < 0.05 at 1, 2, and 3 h), which resulted in a
greater absorption rate for 111In-Cat-Lyz (0.46% dose/h)
(Table 2). On the other hand, the
absorption of 111In-An-Lyz was much slower than that of the
cationic derivatives, although the difference was not significant. The
rates of 111In-Gal-Lyz and 111In-Glc-Lyz were
calculated and found to be comparable with that of
111In-Lyz, indicating that glycosylation has no effect on
the intestinal absorption of Lyz.
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Fig. 6.
Intestinal absorption time courses of
111In-Lyz derivatives calculated by a deconvolution method.
, 111In-Lyz; ,
111In-An-Lyz; , 111In-Cat-Lyz;
, 111In-Glc-Lyz; ,
111In-Gal-Lyz. * P < 0.05, statistically significant difference from 111In-Lyz.
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Table 2.
Intestinal absorption rate of 111In-Lyz and
111In-SOD derivatives following intrajejunal administration
to rats calculated by a deconvolution method
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Intestinal absorption of 111In-SOD derivatives.
Table 1 shows the physicochemical characteristics of SOD and Cat-SOD.
After intrajejunal administration of 111In-SOD and
111In-Cat-SOD, low but significant radioactivity was
detected in plasma (data not shown). However, in the case of
111In-An-SOD, there was no detectable radioactivity in
plasma throughout the experiment that lasted 3 h. Figure
7 shows the intestinal absorption-time
courses of 111In-SOD and 111In-Cat-SOD.
Although 111In-Cat-SOD tended to be more absorbed from the
intestine than 111In-SOD, the amount absorbed was not
significantly different except for the first time point
(P < 0.05). The absorption rate calculated for
111In-Cat-SOD was twofold greater than that for
111In-SOD (Table 2), but these values were less than those
exhibited by the 111In-Lyz derivatives, probably reflecting
the differences in the properties of SOD and Lyz, e.g., the molecular
size (32,000 SOD; 14,300 Lyz).
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Fig. 7.
Intestinal absorption time courses of
111In-SOD and 111In-Cat-SOD calculated by a
deconvolution method. , 111In-SOD;
, 111In-Cat-SOD. * P < 0.05, statistically significant difference from 111In-Lyz.
SOD, superoxide dismutase.
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DISCUSSION |
Cationization is a universal approach applied to increase the
interaction of compounds with negatively charged biological components.
Since Felgner (8) reported efficient gene expression using
cationic lipids for the transfection of cells with plasmid DNA,
cationic molecule-based delivery systems for plasmid DNA have been
extensively investigated in an attempt to achieve nonviral gene
transfer to target cells (33). In addition, enzymes such as SOD (27, 28, 40, 48), glucose oxidase
(21), and catalase (21, 43), as well as serum
albumins (32), immunoglobulins (52), and
ferritin (7), all of which are negatively charged at
physiological pH, have been directly modified with diamines to obtain
cationized derivatives. Cationized proteins exhibit increased cellular
uptake by brain microvascular endothelial cells, hepatocytes, kidney
epithelial cells, and enterocytes. On interaction with the negatively
charged surface of cells, cationized proteins are believed to be
endocytosed or transcytosed through an adsorptive endocytosis/transcytosis process. However, to our knowledge, there have
been few studies to examine the effect of the electrical charge of a
protein on its intestinal absorption. Intestinal epithelial cells
possess a negatively charged surface like other cells. Therefore, in
the present study, the effect of the physicochemical properties, especially the electrical charge, of the protein on its intestinal absorption was explored using an in situ closed loop of rat jejunum. In
this system, the pH of the solution in the loop could hardly change
from the initial value of pH 6.5 during experiment, and the local
luminal pH might not be so different from the pH value of the bulk
solution (22).
A reliable detection method is required to evaluate the intestinal
absorption of proteins. We carried out 111In labeling using
DTPA anhydride to monitor the disposition of proteins because of the
better stability of these radiolabeled proteins compared with their
iodinated counterparts (13, 36). A possible radioactive
metabolite, 111In-DTPA-lysine (9), has only a
limited capacity to cross biological membranes and to escape from cells
where the labeled protein is degraded after endocytosis. When Lyz was
radioiodinated and administered to the rat jejunal loop, the
concentration of trichloroacetic acid-precipitable 125I
radioactivity in plasma was higher than that of 111In
radioactivity after the administration of 111In-Lyz (data
not shown). When radioiodinated Lyz was used for experiments, the
concentration of radioactivity in plasma was much greater than one
obtained with 111In counterpart (P < 0.01). Finding no significant differences between the lytic activity of
Lyz and 111In radioactivity strongly supports the idea that
the transport of intact Lyz derivatives through the intestinal
epithelium can be assayed by monitoring 111In
radioactivity. These considerations suggest that radioiodination might
overestimate the intestinal absorption of proteins, although the reason
for the discrepancy needs to be understood.
Chemical modification greatly changed the tissue disposition
characteristics of Lyz after intravenous injection. As summarized in
our reviews (14, 46, 50), the tissue disposition of
macromolecules is determined mainly by the overall physicochemical
properties, such as the electrical charge and molecular weight, as well
as by the structure involved in the specific recognition, such as monoclonal antibody, lectin, and sugar. Macromolecules having a
molecular size smaller than the threshold of the glomerular filtration
of the kidney are easily filtered and then reabsorbed at the proximal
tubules depending on their property (26). This is the case
for Lyz and SOD, and the cationic nature of Lyz increases the
susceptibility to glomerular filtration and reabsorption. When
111In-Lyz was injected intravenously, the radioactivity was
mainly recovered in the kidney and urine (Fig. 2).
111In-Cat-Lyz, which has about fourfold greater mobility to
the negative pole than Lyz, showed a relatively high accumulation in
the liver, like other cationic proteins, such as cationized bovine
serum albumin (32). No detectable oligomers formed during
the cationization were found during SDS-PAGE (data not shown). On the
other hand, succinylation of Lyz greatly altered the balance of
radioactivity in the kidney and urine. Because the renal uptake of
macromolecules occurs mainly from the luminal side of epithelial cells
(26) and not from the capillary side, such changes
indicate that the reabsorption of Lyz is inhibited by succinylation.
Both 111In-glycosylated Lyz derivatives showed some hepatic
uptake after systemic administration. Hepatocytes are known to possess
asialoglycoprotein receptors on their surface that recognize compounds
having galactose or N-acetylgalactosamine at the nonreducing
end of the sugar chain (1). Synthetically galactosylated
macromolecules are ligands for the receptor (34), and this
is the reason for the high hepatic uptake of 111In-Gal-Lyz.
Although asialoglycoprotein receptors have a much lower affinity for
glucose than galactose, it binds to proteins modified with
2-imino-2-methoxyethyl 1-thioglucoside as used in the present study.
Therefore, asialoglycoprotein receptors are involved in the hepatic
uptake of 111In-Glc-Lyz (34). Although the
imidination used in this study to synthesize glycosylated Lyz has been
reported not to alter the electrical charge of the protein
(24), their electrophoretic mobility was a little lower
than that of Lyz, suggesting a reduced positive charge after
glycosylation. These glycosylated derivatives were prepared because:
1) these modified derivatives possess a positive charge
intermediate between unmodified Lyz and An-Lyz and 2) some
reports have suggested the involvement of specific mechanisms for
sugars associated with enterocytes as far as the transport of
glycosylated macromolecules is concerned (12, 23).
The tissue disposition profile of radioactivity after intrajejunal
administration of each 111In-Lyz derivative was comparable
with that after intravenous administration (Figs. 1 and 2), suggesting
that Lyz derivatives entering the systemic circulation from the
intestine possess unique physicochemical properties. Tissue
uptake clearances, which can be calculated based on the area
under the plasma concentration-time curve and the amount in tissue
(35), were also comparable after intravenous and
intrajejunal administration (data not shown). These results indicate
that the radioactive molecules appearing in the plasma maintain the
structures that determine their tissue disposition.
There have been reports showing that some proteins can be absorbed from
the intestinal tract, although skepticism about the experimental
evidence of transmucosal absorption of proteins is common because of
the limitations of the methodology used (44). Recently,
Castell et al. (5) clearly showed that bromelain, a basic
protein with a molecular mass of 24-26 kDa isolated from the stem
of the pineapple plant, can be absorbed from the intestinal tract of
healthy volunteers as an immunoreactive form of unchanged molecular
mass. The consistency of their results indicates that the absorption of
a certain protein molecule from the gastrointestinal tract is probably
a common phenomenon in healthy adults. As discussed above, the cationic
nature of this protein could help its intestinal absorption, although
the route of its absorption, i.e., transcellular or paracellular, needs
to be identified.
Cationization of Lyz did increase the intestinal absorption of Lyz,
probably through the increase in the isoelectrical point of the enzyme
whose positive charges result in an electrostatic attraction to the
negatively charged proteoglycans of the cell surface, a process that
can lead to adherence to the oppositely charged surfaces
(4). Interaction of proteins with the intestinal tissues
was facilitated by cationization (21, 40). Because binding
to the surface can be considered as the first step in the intestinal
transport of proteins, cationization might be one possible approach to
increase the permeability of proteins. Increased binding of Cat-Lyz was
detected in the specimens of intestinal tissues treated with
FITC-labeled Lyz derivatives.
Recently, glycosylation has been applied to peptides to increase their
transport across the intestinal tissues via a Na+/glucose
cotransporter (30), as well as to produce increased stability to peptidases (29). Although some reports have
suggested involvement of these mechanisms in the transport of
glycosylated macromolecules (12, 23), no improved
absorption was observed for glycosylated Lyz derivatives. Kim et al.
(19) reported that bile acid-conjugated small peptides
could bind to intestinal bile acid transporters without being transported.
The amount of 111In-Cat-Lyz absorbed was significantly
greater than that of 111In-Lyz (P < 0.05 at 1, 2, and 3 h), whereas that of 111In-An-Lyz was
smaller. The absorption rate of Lyz derivatives was proportional to
their electrophoretic mobility (Fig. 8).
These results clearly indicate that the electrical charge of Lyz
determines its intestinal absorption from the jejunal loop. The
apparent permeability coefficient of 111In-Cat-Lyz
(0.135 × 106 cm/s, Table 2), however, was still
much smaller than that of small molecules such as a vasopressin
derivative (43 × 106 cm/s, molecular weight of
1,069) and inulin (4 × 106 cm/s, molecular weight
of 5,200) (37). The relationship between the electrical
charge and intestinal absorption could also be applied to other
proteins such as SOD. 111In-Cat-SOD tended to be absorbed
faster than 111In-SOD, although the amount absorbed was not
significantly different at later time points. With these protein
derivatives, it is quite obvious that the molecular weight is a very
important factor determining their intestinal absorption. Although
111In-Cat-SOD had a greater electrophoretic mobility to the
negative pole than 111In-Lyz, its absorption rate was
smaller than that of 111In-Lyz, showing the importance of
the molecular weight of the protein as far as its intestinal absorption
is concerned. It is difficult to conclude that these protein
derivatives are absorbed through the transcellular or paracellular
route. The absorption through the both routes could be enhanced by
cationization (10, 42). Further studies are needed to
clarify the contribution of each route to the intestinal absorption of
the derivatives.
View larger version (16K):
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|
Fig. 8.
Dependence of the intestinal absorption rate of
111In-Lyz and 111In-SOD derivatives on the
electrophoretic mobility measured by a zeta sizer. ,
111In-Lyz derivatives; ,
111In-SOD derivatives. The absorption rate of a derivative
calculated by the deconvolution method is plotted against its
electrophoretic mobility.
|
|
In conclusion, it has been shown that the intestinal absorption of a
protein is regulated by its electrical charge, if the molecular size of
protein is not altered. Enhanced adsorption of a cationic derivative to
the surface of the tissue would result in its increased permeability
through the barrier. These findings provide useful information about
the intestinal absorption of proteins of immunological and/or
pharmacological importance in normal, healthy subjects.
| |
ACKNOWLEDGEMENTS |
This work is supported, in part, by a grant-in-aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: M. Hashida, Dept. of Drug Delivery Research, Graduate School of
Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan (E-mail: hashidam{at}pharm.kyoto-u.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00358.2001
Received 10 August 2001; accepted in final form 12 December 2001.
| |
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