Intestinal Uptake oÃ-Fatty Acids Complexed to

Proteins in the Chick Intestine

D. SKLAN ANDS. HURWITZ
Faculty of Agriculture, Hebrew University of Jerusalem,
Rehovot, Israel

ABSTRACT Intestinal mucosal uptake of protein complexed-fatty acids

was studied in ligated duodenal loops in the chick. Increasing the concen
tration of an albumin oleic acid-complex resulted in a linear increase in
uptake of oleic acid. Varying the albumin-to-oleic-acid ratio with constant
albumin concentration resulted in depressed oleic acid uptake when the
ratio was below 1:3. Uptake of oleic acid complexed to albumin was in
creased by some 60% on addition of taurocholic acid above its critical
micellar concentration. In the absence of albumin, oleic acid uptake was
some 60% high from a micellar solution. Uptake of lauric acid from
aqueous solution was linear with concentration until its maximum solubility
was reached, whereas uptake from albumin complexes at varying lauric
acid concentrations was not linear with increasing concentration. Stearic
acid exhibited lowest uptake and linoleic and linolenic acid highest uptake
both when complexed to albumin or from micellar solution, although
albumin-complexed fatty acids were transported at about half the rate of
micellar fatty acids. We concluded that some proportion of fatty acids com
plexed to lipophilic proteins can be absorbed in the intestine in the absence
of bile acids. When oleic acid was complexed to casein, bovine serum
albumin or /8-lactoglobulin at a protein :oleic acid ratio of 1:10 serosal
transport was 40 to 50% of mucosal uptake. J. Nutr. 110: 270-274, 1980.
INDEXING KEY WORDS fatty acid • absorption • chick •
intestine •protein complexes

Absorption of lipids in the small intestine
is preceded by hydrolysis of dietary tri-
glycerides to monoglycerides and free fatty
acids, and formation of mixed micelles with
bile salts and phospholipids (1, 2). The
rate-determining step in fatty acid absorp
tion from micelles appeared to be the rate
of penetration of the lipid through the un-
stirred water layer adherent to the mucosal
brush border membrane (3) when a syn-
thetic bile acid-fatty acid system was used,
We have recently shown that the presence
of lipophilic proteins depressed the uptake
of both fatty and bile acids from ligated
intestinal segments, presumably due to
binding of fatty acids and bile acids to the
protein (4). However, in vitro intestinal
uptake of fatty acids complexed to
min occurred, but was some threefold
lower than that measured with micellar
fatty acids ( 1 ). The purpose of this study
was to further elucidate the role of protein
in intestinal uptake of fatty acids,
MATERIALSAND METHODS
We commercially obtained radioactive
compounds ( Radiochemical Centre, Amer-
sham, England) and bovine serum, albu-
min ( fatty acid-free ), casein ( vitamin-free)
and ¿8-lactoglobulin( Sigma Chemical Co.,
St. Louis, MO).
The animals used were 3 to 4-week old
Leghorn-X-Hampshire male chicks. The
chicks were anesthetized with sodium
_____
albu- Received for publication 12 June 1979.

270
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 FATTY ACID ABSORPTION FROM PROTEIN COMPLEXES 271

pentobarbital and the duodenum exposed TABLE 1

with nervous and vascular systems intact. Mucosal uptake and seroso/ transport of oleic acid
The duodenum was washed with 0.9% (OA) complexed to various proteins from the
NaCI at 37°and ligated at the appropriate duodenum of the chick1
sites. One half milliliter of the test solution
containing labeled fatty acid and non- ProteinAlbumin uptakenmoles1.99
transfer•cm"1
absorbed marker was introduced into the
segment and after 10 minutes, chicks were -min"10.91
killed with an intracardiac overdose of ±0.24 ±0.09«
sodium pentobarbital. The contents of the Casein 2.03±0.26 0.79±0.08°''
/3-lactoglobulinMucosal 1.84±0.12Serosal 0.69±0.096
ligated segment were flushed with 0.9%
NaCI at 4°and brought to volume with
1Thirty nmoles of protein and 300 nmoles of OA
the same solution. Duplicate aliquots of were added per 0.5 ml. Mean±so of six chicks.
these and unincubated test solutions were Values in columns not followed by same superscript
lyophilized for radioactivity determina differed significantly (P < 0.05).
tions. The length of the segment was de
termined at the end of the incubation RESULTS
period. Mucosal uptake and serosal transfer of
Tissue samples were homogenized in a oleic acid (OA) complexed to bovine
highspeed blender, brought to volume and serum albumin, casein and 0-lactogIobulin
duplicate aliquots lyophilized for radio are shown in table 1. Mucosal uptake was
activity determinations. Complexes of fatty similar when oleic acid was complexed to
acids, 14C-labeled fatty acids and proteins all of the three proteins. At the pro
were prepared as described by Peters et al. tein :oleic acid ratio of 1:10 used here,
(5). Test solutions were prepared by trans serosal transfer was 40 to 50% of the mu
ferring known amounts of protein-fatty cosal uptake with uptake from albumin-
acid complexes to bicarbonate buffer at pH complexed oleic acid higher than uptake
6.5, and adding trace amounts of 3H-dex- from /î-lactoglobulin-complexed oleic acid.
tran as non-absorbed marker (6). The Increasing the concentration of a 1:6
solution was gassed with 5% CO2 in O2, albumin :oleic acid complex yielded a
held overnight at 37°and regassed before
linear increase in mucosal uptake (fig. 1).
use. No significant effect on uptake was ob
Radioactivity was determined by liquid served on varying the pH of the buffer con
scintillation, with samples counted simul taining the albumin-oleic acid complex
taneously in two channels. Contribution of from 6.0 to 7.5 (not shown).
each isotope in the channel of the other Uptake of oleic acid from albumin-oleic
and quench correction were calculated acid complexes was determined at constant
using internal standards of both isotopes. albumin and varying oleic acid concentra
Calculations. The unidirectional mucosal tions (table 2). Fractional mucosal uptake
uptake was calculated from the change in was lower at albumin :oleic acid ratios at
ratio of the probe molecule to the non- or below 1:3 than at higher ratios. In ad
absorbed dextran marker and uptake ex dition, the effect of the addition of tauro-
pressed as nmoles-miiT'-crrr1, and frac cholic acid on uptake of oleic acid from
tional uptake as MP-min^-cnr1. Serosal albumin complexes is also shown in table
transfer was taken to be the difference be 2. Addition of 1 mM taurocholic acid did
tween the amount of label taken up by the not affect uptake, whereas, addition of 8.0
mucosa as described above and the amount mM enhances oleic acid uptake. For fur
of label remaining in the intestinal tissue ther comparison, uptake of oleic acid-
and is likewise expressed as nmoles •
min'1 •
taurocholic acid micelles, in the absence of
cm'1.
albumin, resulted in greater oleic acid up
Analysis of variance and deviation from take which was more than twofold that
linear regression were calculated using observed from albumin complex in the ab
standard methods. sence of taurocholic acid.

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 272 D. SKLAN AND S. HURWITZ
1§-i
1 albumin in the presence or absence of
6uvES0ECï21
-500
1 I , ,
0 ¿00 800
CONCENTRATION (nmoles)
Fig. l Mucosal uptake of oleic acid at vary
ing concentrations of an albumin-oleic acid com
plex from chick duodenal loops. The albumin:oleic
acid ratio was 1:10 and each point represents the
mean ±so of five chicks.
Mucosal uptake of lauric acid was deter
mined both from buffer in the absence of
albumin and as a complex with constant
albumin and varying lauric acid concentra
tion. The fractional mucosal uptake of
lauric acid at varying concentrations from
aqueous solution was constant until the
solubility limit of lauric acid was reached
(3). Uptake from the albumin complex
was not linear with lauric acid concentra
tion and was depressed at albumin :lauric
acid ratios below 1:2.2 (F < 0.05), and
was some 20% lower than uptake from
aqueous solution (P < 0.05). At concentra
tions of the lauric acid-albumin complex
above the maximum, aqueous solubility
fractional uptake was similar to that ob
served from aqueous solution. Results are
presented in figure 2 as the log-log plot,
and uptake from aqueous solution is linear
with increasing concentration, but uptake
from albumin complex showed deviations
from linearity ( P < 0.05 ) at low albumin
to lauric acid ratios.
Rates of mucosal uptake were deter
mined for several fatty acids complexed to
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taurocholate (table 3). In the absence of
taurocholate, uptake of stearic acid was
similar to that of palmitic and lower than
that of unsaturated C18 fatty acids. Addi
tion of the taurocholate to the albumin-
fatty acid, increased uptake of all fatty
acids although uptake of stearic and pal
mitic acids remained lower than uptake of
oleic, linoleic and linolenic acids.
DISCUSSION
We have recently shown (4) that addi
tion of lipophilic proteins to taurocholic
acid-oleic acid micelles reduced mucosal
uptake of both the fatty and bile acid,
probably due to binding to the protein.
However, in addition, we found that fatty
acids complexed to three different lipo
philic proteins are both taken up and trans
ported by the intestinal mucosa. Serosal
transport in the duodenal segments was
40 to 50% of the mucosal uptake, which
was similar to values obtained in a pre
vious study with micellar oleic acid in the
rat jejunum (7). Thus, bile salts are not
essential for the transport of fatty acids.
This may explain the 20-50% absorption of
lipids observed in chicks with ligated bile
ducts (8).
Increasing the concentration of a given
complex of oleic acid with albumin in
ligated segments resulted in a linear in-
TABLE 2
Effect of varying the ratios of oleic add (OA ) to
albumin on mucosal uptake from the
duodenum of the chick'
uptake10f-cm~l-min~l1.462
OAnmoles180360180904510180180Ratio
Alb:OA1:121:61:31:1.51:0.31:61:6Taurocholic
acidtimóles4—————0.54Fractional
±0.120»0.641
±0.044"0.634±0.05160.541
±0.044'0.471
±0.056'0.510±0.051r0.662
±0.073'1.046±0.071-<
1Mean±SDof five chicks. Albumin was present
at constant levels of 30 nmoles/0.5 ml. Values in
columns not followed by the same letter differ sig
nificantly (P < 0.05).
 FATTY ACID ABSORPTION FROM PROTEIN COMPLEXES
crease in mucosal uptake, without any sign
of saturation. It seems reasonable to as
sume that under these conditions, uptake of
oleic acid was by diffusion as previously
observed in micellar systems in the proxi
mal small intestine (9).
Varying the protein-to-fatty-acid ratio
was carried out with lauric acid and was
compared to the uptake from the aqueous
system. The log-log plot would be expected
to be linear with slope unity if uptake is
by diffusion at a single constant rate and
the intercept proportional to the rate con
stant. This was the case with the uptake
of lauric acid from aqueous solution but is
not so when the lauric acid was complexed
to albumin, where at low-lauric-acid-to-
albumin ratios uptake rate was depressed.
While lauric acid may bind to albumin or
be taken up by the intestine in a manner
different to long chain fatty acids, similar
results were obtained on examining the up
take of oleic acid at differing albumin :OA
ratios. At OA: albumin ratios below 1:3,
0123
log CONCENTRATION (jumóles)
Fig. 2 Log-log plot of mucosal uptake of
lauric acid from buffer ( •) and from albumin
complexes (30 nmoles albumin/0.5 ml, O) against
lauric acid concentration from chick duodenal
loops. Results represent means ±so where these
do not fall within the circles of five chicks. Lauric
acid uptake from albumin complexes was signifi
cantly depressed ( P < 0.05 ) as compared to up
take from aqueous solution.
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273
TABLE 3
Uptake of different fatty acids complexed to albumin
from chick duodenum: effect of addition of
taurocholic acid (TC)1
UptakeFatty
albumin
acidLauric andTCcm~l
albuminnmoles
•1.226±0.095° mm"11.448±0.268a'1'
•
0.819±0.1296'c 1.420±0.174"
Palmitic
Stearic 0.646±0.072« 1.271±0.145°
Oleic 1.141Õ0.081"-11 1.880±0.1316
Linoleic 1.171Õ0.106"'" 2.337±0.221»
LinolenicWith 1.181±0.032°'6With
2.107±0.113"
1Thirty nmoles albumin and 180 nmoles fatty
acids were present per 0.5 ml test solution. Results
are mean±so of five chicks. Values in columns not
followed by the same letter differ significantly
(P < 0.05).
uptake was reduced as compared to higher
ratios. Based on the Scatchard binding
model, bovine serum albumin exhibits three
different binding sites, where the primary
site appears to bind three moles of fatty
acid, the secondary sites, three moles, with
tertiary sites binding some 20 or more
moles (10). It is tempting to speculate
that the strongly bound fatty acids at the
primary sites are less available for absorp
tion than those bound to the secondary or
tertiary sites. Uptake of albumin-com-
plexed fatty acids may speculatively not
be limited by the rate of diffusion through
the unstirred water layers but by the rate
of dissociation of the protein-fatty acid
complex close to the brush border.
Addition of bile acids below their criti
cal micellar concentration did not alter
uptake of albumin-complexed fatty acids,
whereas addition above the critical mi-
cellar concentration enhanced fatty acid
uptake presumably as micelles were then
formed. Presence of albumin resulted in
lower uptake of fatty acids from a bile
acid-fatty acid system as previously re
ported (4).
Comparison of the mucosal uptake of
different fatty acids from albumin com
plexes revealed that stearic acid uptake
was lower than that of the more unsatu-
rated oleic, linoleic and linolenic acids.
 274 D. SKLAN AND S. HURWITZ
Little differences are apparent in the values
of the binding constants of the different
fatty acids to albumin ( 10), and thus, dif
ferences in the rate of uptake may stem
from differences in membrane permeability
or affinity for the fatty acid binding pro
teins of the mucosal cells (11). Addition
of bile salts above the critical micellar con
centration considerably enhances uptake
of all fatty acids tested, with palmitic and
stearic acids exhibiting lowest uptake and
polyunsaturated fatty acids highest uptake.
This parallels the overall percentage up
take of dietary fatty acids previously re
ported in the chicken ( 12, 13) and also
micellar solubility (14); both in the pres
ence and absence of bile acids, stearic acid
shows poorest uptake and linoleic and
linolenic acid, highest uptake.
The action of lipophilic proteins with
respect to intestinal fatty acid uptake may
be tentatively summed up as follows: one
process would involve binding of fatty
acids and bile acids, thus reducing the
concentration in micellar solution, hence,
reducing their rate of uptake. A second
process involves solubilization of fatty acids
by the protein transport to the brush
border and uptake of the fatty acid after
dissociation from the protein. The latter
process of fatty acid uptake was some
threefold slower than uptake from micelles
as was previously observed in vitro (1).
ACKNOWLEDGMENT
The skilled technical assistance of Mrs.
O. Kedar is gratefully acknowledged.
LITERATURE CITED
1. Johnston, J. M. (1963) Recent develop
ments in the mechanism of fat absorption. In:
Advances in Lipid Research (Paoletti, R. &
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Kritchevsky, D., eds.), Vol. 1, pp. 105-133.
2. Senior, J. R. (1964) Intestinal absorption
of fats. J. Lipid Res. 5, 495-521.
3. Westergaard, H. & Dietschy, J. M. (1976)
The mechanism whereby bile acid micelles
increase the rate of fatty acid and cholesterol
uptake into the intestinal mucosal cell. J. Clin.
Invest. 58, 97-108.
4. Sklan, D., Budowski, P. & Hurwitz, S. (1979)
Absorption of oleic and taurocholic acid from
the intestine of the chick: interference by
proteins. Biochim. Biophys. Acta 573, 31-39.
5. Peters, T., Tanuichi, H. & Anfinsen, C. B.
(1973) Affinity chromatography of serum al
bumin with fatty acids immobilized on aga-
rose. J. Biol. Chem. 248, 2447-2551.
6. Sallee, V. L., Wilson, F. A. & Dietschy, J.
M. (1972) Determination of unidirectional
uptake rates for lipids across the brush border.
J. Lipid Res. 13, 184-192.
7. Sklan, D. & Budowski, P. ( 1977 ) Hydroly
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intestine of the chick. Lipids 12, 193-197.
8. Garrett, R. L. & Young, R. J. (1975) Effect
of micelle formation on the absorption of
neutral fat and fatty acids by the chicken. J.
Nutr. 105, 827-838.
9. Schiff, E. R., Small, N. C. & Dietschy, J. M.
( 1973 ) Characterization of the kinetics of the
passive and active transport mechanisms for
bile acid absorption in the small intestine and
colon of the rat. J. Clin. Invest. 51, 1355-1361.
10. Spector, A. A. ( 1975) Fatty acid binding to
plasma albumin. J. Lipid Res. 16, 165-179.
11. Ockner, R. K., Pittman, J. P. & Yager, J. L.
(1972) Differences in the intestinal absorp
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fatty acids. Gastroent. 62, 981-992.
12. Renner, R. & Hill, F. W. (1960) Utilization
of com oil, lard and tallow by chickens of
various ages. Poult. Sci. 39, 849-854.
13. Sklan, D., Budowski, P., Ascarelli, I. & Hur-
witz, S. (1973) Lipid absorption and secre
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J. Nutr. 103, 1299-1305.
14. Hoffman, A. F. & Borgstrom, B. (1962)
Physico-chemical state of lipids in intestinal
contents during their digestion and absorp
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