Inhibition of cholesterol absorption in rats by plant sterols

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					       Inhibition of cholesterol absorption in rats by plant
                        Ikuo Ikeda,' Kazunari Tanaka,2 Michihim Sugano, George V. Vahouny, and Linda L.
                         Department of Biochemistry, The George Washington University, School of Medicine and Health
                         Sciences, Washington, DC 20037

Abrrtract The extent and site(s) of inhibition of cholesterol ab-          absorption, by contrast, does not inhibit the lymphatic ab-
sorption by plant sterols, sitosterol and hcosterol, were studied in       sorption of endogenous cholesterol (5).
rats. The intragastric administration of a single emulsified lipid            It has been suggested that these structural analogs func-
meal containing 25 mg [5H]cholesteroland 25 mg of either sitos-
terol or hcosterol inhibited the lymphatic absorption of cholesterol
                                                                           tion (1-3) by competing with cholesterol at steps essential
by 57% and 41%, respectively, in 24 hr. Less than 2% of each               for absorption including: micellar solubilization, uptake by
plant sterol was absorbed in the 24-hr period. In contrast, neither        the brush border membrane, intracellular esterification,

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plant sterol (50phi) inhibited cholesterol absorption when co-ad-          andlor incorporation into chylomicrons. With respect to
ministered with equimolar amounts of cholesterol in phospholipid-          lumenal events, cholesterol and sitosterol, as well as their
bile salt micelles nor was either absorbed from the micellar solu-
tion. A series of in vitro studies was conducted to identify the site(s)
                                                                           7-dehydro derivatives (7), are reported to bind to isolated
of plant sterol inhibition of cholesterol absorption and to account        rat brush border membranes (7), to the brush border mem-
for the difference in inhibitory effectiveness of sitosterol and           branes of either rat jejunal loops in situ (8) or isolated rat
fucosterol. A comparison of the micellar solubility of each sterol                               7,
                                                                           jejunal villus cells ( ) and to brush border isolated from
alone and in equimolar binary mixtures (to 2.0 mM) revealed that           rat proximal small intestine 2 hr after the in vivo adminis-
the Solubility of individual sterols decreased in the following order:
                                                                           tration of each sterol in a lipid meal (9). In each case, less
cholesterol, fucosterol, sitosterol, and that in binary mixtures
cholesterol solubility was decreased by sitosterol and, to a lesser        sitosterol associates with this membrane. Nonetheless, if
extent, by fucosterol relative to its solubility alone. A comparison       sitosterol were present in high doses, it might inhibit cho-
between micellar-solubilized cholesterol and either sitosterol or          lesterol binding. Another major lumenal event, micellar
fucosterol for binding to isolated brush border membranes, in-             solubilization of sterols, represents a likely site for sterol
testinal mucin, or for esterification by either cholesterol esterase
or acyl coenzyme Acholesterol acyltransferase revealed moderate            competition. Related to this, a recent study (10) suggests
to no competition. The data suggest that plant sterols displace            that sitosterol has a greater affinity than cholesterol for
cholesterol from bile salt (taurocholate) micelles and that sitosterol     taurocholate micelles and would displace micellar choles-
is more effective than fucosterol in this capacity.-Ikeda, I.,             terol with a favorable free energy change.
K.Tan*,       M.Sugano, G. V. Vahouny, and L. L. Gallo. Inhi-                 With respect to intracellular steps, the amount of plant
bition of cholesterol absorption in rats by plant sterols. J. L p dii
Rw. 1988. 29: 1573-1582.                                                   sterol taken up by intestinal cells may be insufficient to ac-
                                                                           count for significant inhibition of cholesterol processing,
Supplemmtarykeywords sitosterol hasterol hypocholesterolemia               i.e., esterification or incorporation into chylomicrons. In
  brush boder stem1 esterification mucin sterol solubility miaelles        this regard Swell et al. (11) reported that sitosterol was taken
  bile salts lymph cannula

   Plant sterols are effective inhibitors of cholesterol absorp-             Abbreviations: C, cholesterol; S, sitoaterol; F, hasterol; BSA, bovine
tion, i.e., they display hypocholesterolemic properties (1-3).             serum albumin; DTNB, 5,Y-dithiobis(2-nitrobenzoicacid); ACAT, acyl
From a structural standpoint, this inhibition is related most              coenzymeAcholesterol ay-c l,           F,C phosphatidylcholine;PE, phos-
                                                                           phatidylethanolamine;MO, monoolein, OA, oleic acid, BS, bile salt; TLC,
clearly to substitutions in position 24 (1-6) on the sterol                thin-layer chromatography; GLC, gas-liquid chromatography.
side chain as demonstrated with sitosterol (24-ethyl cho-                     'Present addrew Laboratory of Nutrition Chemhy, Kyuahu U i e s t ,
lesterol), stigmasterol (622,24-ethyl cholesterol), sitostanol             School of Agriculture, 46-09, Hakasaki 6-10-1. Higashi-ku,Fukuoka 812,
(5-dihydro, 24-ethyl cholesterol), and campesterol (24-                    Japan.
                                                                              %tsentaddress: Laboratoxy of Nutrition, Nagasaki PrCfectxral Women's
methyl cholesterol). Fucosterol(24-ethylidene cholesterol),                Junior College, Narutaki 1-4-1, Nagasaki 850, Japan.
although untested as an inhibitor of dietary cholesterol                      'To whom reprint requests should be addressed.

                                                                                 Journal of Lipid h e a r c h      Volume 29, 1988          1573
up by the intestinal wall, but Borgstriim (12) did not observe   All animals post-surgery were allowed free access to drink-
any accumulation of administered sitosterol and our recent       ing water containing 5% glucose and 0.9% NaC1; those rats
observations support the latter finding (9). Moreover, it has    that would later receive the emulsified lipid meals were in-
been reported that microsomal sitosterol does not compete        fused intragastrically with this same solution at a rate of
with cholesterol for ACAT-catalyzed esterification (13).         3.0 ml/hr. Animals with bile diversion received, instead, a
   The present studies represent a systematic approach to        continuous infusion of “artificial”bile (preparation described
examining the relative inhibitory effect of sitosterol and       below). This was infused intraduodenally at a rate of 3.4 mll
fucosterol (plant sterols with ethyl and ethylidene substi-      hr from the time of surgery to killing with a brief interrup-
tutions at carbon 24, respectively) on the lymphatic absorp-     tion for intraduodenal administration of the radiolabeled
tion of exogenous cholesterol and to accounting for this in-     micellar sterol(s). Animals with lymph flow rates of > 2.0
hibition with in vitro studies that examine I) the micellar      mlfhr were given the lipid meals as described in the indi-
solubility of each plant sterol and the effect of each on the    vidual studies between 9 and 10 the morning after surgery.
micellar solubility of cholesterol, and 2) the competition
between plant sterols and cholesterol for binding to brush       Preparation of lipid meals and ”artifxid’’ bile for in vivo
border membranes, for binding to mucin, and for esterifica-      sterol absorption studies
tion by mucosal cholesterol esterase and ACAT.                      Lipid emulsions for intragastric administration were pre-
                                                                 pared as described earlier (5) and contained per 3 ml of
                                                                 physiologic saline: 50 mg BSA, 292 mg oleic acid, 279 mg
                                                                 sodium taurocholate, and 25 mg of [3H]cholesteroleither
                                                                 alone or plus 25 mg of sitosterol or fucosterol. These emul-

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                                                                 sions were prepared immediately before use and re-
Materials                                                        homogenized before administration.
   Bovine serum albumin (BSA) fraction V (fatty acid-               Sterol-containing micellar solutions were prepared for
poor), ATP, coenzyme A, monoolein, oleic acid ( > 99%            intraduodenal infusion and contained 6.6 mM sodium
purity), fucosterol (>95% purity), and egg yolk phos-            taurocholate, 0.6 mM egg yolk phospholipids, and 50 gM
pholipids (Type IX-E) were obtained from Sigma Chem-             [3H]cholesteroleither alone or plus 50 gM of [4-’4C]sitos-
ical Co., St. Louis, MO. Cholesterol (>99% purity) was           terol or fucosterol in 15 mM sodium phosphate buffer, pH
purchased from Serdary Research Laboratories,                    7.4, containing 62 mM NaCl and 2.5% glucose. The so-
Ontario, Canada. Sitosterol (from ICN Pharmaceuticals,           lutions were sonicated, filtered through ACRODISC (0.2
Cleveland, OH) was recrystallized from ethyl acetate-            pm, Gelman Sciences, Inc., Ann Arbor, MI), and kept at
methanol to >98% purity. Sodium taurocholate (> 96%              37OC. These preparations were used as described in the
purity) was from Calbiochem, LaJolla, CA. [l-’4C]Oleic           individual studies.
acid, [4-’4C]cholesterol and [lar,2a-3H]cholesterol and             “Artificial”bile for intraduodenal infusion into bile-fistula
[4-’4C]sitosterolwere supplied by Amersham, Arlington            rats was prepared as a micellar solution containing 6.6 mM
Heights, IL. All other chemicals were reagent grade and          sodium taurocholate and 0.6 mM egg yolk phospholipids
purchased from Fisher Scientific, Silver Spring, MD.             with no sterol, with 150 pM cholesterol, or with 150 pM
                                                                 each of cholesterol and sitosterol in 15 mM sodium phos-
Animals                                                          phate buffer, pH 7.4, containing 62 mM NaCl and 2.5%
   Adult male Wistar rats (Charles River Breeding Labora-        glucose. The procedure for preparation was the same as
tories, Wilmington, MA) weighing 200-240 g were housed           that described above for the micellar sterol meals.
under a normal (12 hr) light cycle and allowed laboratory
chow (Ralston-Purina Co., St. Louis, MO) and water ad            Preparation of micellar solutions for in vitro studies
libitum prior to use.                                               A series of micellar solutions were prepared for the in
                                                                 vitro studies. These varied in the number and type of com-
Surgical procedures                                              ponent, as well as in the concentration of sterol. Each con-
   Animals were anesthetized with sodium pentobarbital           tained 6.6 mM sodium taurocholate and 25 pM to 2.0 mM
(50 mg/kg body weight) and subjected to cannulation of the       sterol (cholesterol, sitosterol, or fucosterol, alone or in bi-
left thoracic lymphatic cephalad to the cisterna chyli as        nary combinations) and where indicated in the individual
previously described (14). A second indwelling catheter was      experiments, 0.6 mM egg yolk phospholipids (67.7 mol %
placed either in the stomach or in the duodenum for later        PC and 22.7 mol % PE based on phosphorus content), 1
administration of either an emulsified or a micellar lipid       mM oleic acid, and 0.5 mM monoolein. These were pre-
meal, respectively. In some studies, where indicated, bile       pared in Hank‘s balanced salts solution containing 15 mM
was drained by an indwelling bile duct catheter without          HEPES, pH 7.4, when used in brush border binding stud-
interruption of pancreatic juice flow. After surgery, animals    ies (to preserve the membrane) or in 15 mM sodium phos-
were placed in restraining cages in a warm recovery room.        phate buffer, pH 7.4, containing 132 mM NaCl when used

1574      Journal of Lipid Research Volume 29, 1988
in the mucin binding and micellar sterol solubility studies.      (7). The brush border membranes were consistently en-
All were sonicated, filtered, and gassed as described above.      riched 20 times in sucrase activity and 15 times in alkaline
                                                                  phosphatase activity relative to the whole homogenate.
Preparation and administrationof the semi-purifieddiet            Recovery of ACAT (19), succinic dehydrogenase (20), and
   A semi-purified diet was prepared (Dyets, Inc., Bethle-        DNA (21), markers for microsomes, mitochondria, and
hem, PA) to contain 20% casein (vitamin-free), 10%                nuclei, respectively, in the brush border was less than 0.5%
safflower ol (plant sterol-free), 4% mineral mixture (Rogers-
           i                                                      of levels in the whole homogenate.
Harper), 1% vitamin mixture (Harper), 4% cellulose, 0.2%
                                                                  Preparation of mucin
choline chloride, and 60.8% sucrose. Sterols were added
at a level of 0.25% at the expense of sucrose. Rats were             Surface material was aspirated under line vacuum from
meal-fed 10 g of the diet over a period of 1 hr for 7 days.       the proximal half of everted small intestine that had been
On the final day of the study, 5 pCi of [3H]cholesterolwas        flushed with 100 ml of iced 0.9% NaCl. Crude mucins were
added to each diet.                                               suspended in iced 0.154 M sodium phosphate buffer, pH
                                                                  7.2, and homogenized for 30 sec in a Potter-Elvehjem tube
Collection of lymph                                               with a motor-driven Teflon pestle. Mucins were isolated
   Lymph was collected at 3, 6, 9, and 24 hr in iced              from the suspension by centrifugation at 4% for 30 min
heparinized tubes containing DTNB (1 mM), an inhibitor            at 6000 g (22) and were diluted with the same buffer to a
of 1ecithin:cholesterol acyltransferase.                          final protein concentration of 10 mg/ml. Mucin enrichment
                                                                  (threefold) in the supernatant was assessed by radioim-
Determination of micellar solubility of sterols in vitro          munoassay (22).

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and in vivo
   In the in vitro studies, 2 ml of each micellar solution con-   Transfer of sterols from micellar solutions to brush
taining sterols singly or in binary combinations (prepared        border membranes and to mucin
as described above with a specific composition as described          In the brush border experiments, 4 ml of the micellar
in the individual experiments) at 37'C was monitored im-          solution (prepared as described above with a specific com-
mediately for turbidity by reading absorbance at 430 nm           position as described in individual experiments) was in-
(Beckman, Acta V spectrophotometer). The micellar so-             cubated with 1 ml of brush border membrane suspension
lutions were maintained at 37OC overnight and observed            (2 mg protein) at 37% (7). At 30 min (representing initial
for precipitates. In a separate study, selected micellar so-      rate of binding), 1-2-ml samples were withdrawn and
lutions (3 ml), as defined in the individual experiments,         released into 5 ml iced 0.9% saline containing 7 mM so-
were centrifuged at forces of 10,000 g up to 100,000 g (Beck-     dium taurocholate, which decreases nonspecific sterol ad-
man L8, 50 Ti rotor). The concentration of each sterol in         sorption. This was centrifuged for 30 min at 27,000 g at
the clear supernatant was determined either by radioac-           5%. The resulting pellet was washed once in 10 ml of the
tivity or mass measurement.                                       same solution and recollected by centrifugation. The brush
   In the in vivo studies, rats were killed 2 hr after con-       border membrane pellet was suspended in 1.0 ml distilled
sumption of the final sterol-containingmeal. Lumenal con-         water, sonicated, and subjected to radioactivity and pro-
tent (2 ml average volume) was collected from each animal,        tein determinations (18). The data were corrected for zero-
heated for 15 min at 70°C to inactivate lipolytic enzymes,        time binding ( < 10 nmol/mg protein).
and centrifuged at 100,000g for 60 min. [3H]Cholesterol              In the mucin experiments, 3 ml of the micellar solution
in the clear micellar solutions was determined by radioac-        (composition as described in individual experiments) was
tivity measurement.                                               incubated with 1.0 ml of mucin suspension (10 mg protein)
                                                                  at 37'C. At 15 min (representing initial rate of binding),
Preparation of brush border membranes                             l-ml samples were withdrawn and released into 5.0 ml of
                                                                  iced 0.3 M sucrose containing 7 mM sodium taurocholate,
  Brush border membranes were prepared from mucosa                mixed, and centrifuged at 5% for 15 min at 100,000g. The
scraped from the proximal half of the small intestine as          mucin pellet was washed once with the same solution and
described by others (15). The membrane pellet was                 again centrifuged. Pellet-associated sterol was determined
resuspended in 5.0 ml of 50 mM mannitol 2 mM HEPES,               by radioactivity counting and pellet protein was assayed
pH 7.1, the preparation buffer. After assay for alkaline phos-    (18). The data were corrected for zero-time binding (< 20
phatase ( 6 ,sucrase (17), and protein (18), the membranes
          1)                                                      nmol/mg protein).
were pelleted by centrifugation for 30 min at 27,000 g and
resuspended in Hank's balanced salts solution containing
15 mM HEPES, pH 7.4, 5 mM EGTA, and 4% BSA (fatty                 Esterification enzyme assays
acid-free) to give a final protein concentration of 2 mg/ml.        Cholesterol esterase activity was assayed as previously
The suspension was gassed with 02-C02 95:5 as suggested           described (23) in cytosol (2.0 mg protein) prepared from a

                                                                    Zkcda et al.   Inhibition of cholesterol absonption   1575
20% homogenate of mucosa scraped from the proximal
small intestine. The conversion of sterol into steryl ester
was measured in the presence of either 0.5 pmol [3H]choles-
terol or [‘4~]sitosteroldone or hthe presence of 0.5 m o l of
both sterols with first one and then the other radiolabeled.                 B
   ACAT activity was assayed as previously described (19)                    e
(but scaled up tenfold) in microsomes (1.5 mg protein) pre-                  5:
pared from a 10% homogenate of mucosa scraped from the                       6
proximal small intestine and preincubated for 30 min at
37OC with either 84 nmol [3H]cholesterolor [4-“C]sitos-
terol, or mixtures containing 84 nmol of both sterols with
first one and then the other radiolabeled. The sterols were                                   €v                           s+c
added in 5 11dioxane-propylene glycol 2:l (v/v) (19). Under
the preincubation conditions described, each sterol alone
and in equimolar concentrations (over a range of 10-175
nmol total sterol added) bound to the same extent and                                          3      6       9                                     24
linearly to microsomal protein (results not shown). For ex-                                                       Time (Hours)
ample, when 175 nmol of cholesterol, sitosterol, or 87.5                     Fig. 2. Effect of micellar sitosterol and fucosterol on micellar cholesterol
nmol of each was preincubated with microsomes, 154 5 8                       absorption. Lymph and bile duct-cannulated rats were intraduodenally
nmol of total sterol was bound.                                              infused for 24 hr with “artificial”bile (3.4 ml/hr). Then 2 ml of micellar

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                                                                             sterol(s), in the concentrations given below, was infused. Lymph was col-
   In both enzyme assays, aliquots were removed from the                     lected with time for 24 hr and analyzed for sterol radioactivity by liquid
digest with time and added to Folch solvent (24) containing                  scintillation counting. Each data point is the mean for n = 4-6; C, 50
[3H]-or [14C]cholesteryl  oleate (20,000 dpm) as an internal                 FM [3H]cholesterol;C + s, 50 gM [sH]cholesterol + 50 /AM[‘*C]sitos-
                                                                             terol; C + F, 50 FM [3H]cholesterol + 50 p M fucosterol.
standard to correct for procedural losses. Free and esterified
sterol were separated by thin-layer chromatography as
previously described (19).                                                   Sterol analyses
                                                                                Sterols were estimated by liquid scintillation counting
                                                                             of radioactivity (19) or by GLC determination of mass (5).
                      C absorption     ------
            I     S of F absorption    -                             1

                                                                             Inhibition of exogenous cholesterol absorption by
                                                                             sitosterol and fucosterol
                                                                                The lymphatic absorption of cholesterol alone and in the
                                                                             presence of plant sterols was followed for 24 hr after ad-
                                                                             ministration in an intragastric meal. As shown in Fig. 1,
                                                                             the absorption of exogenous cholesterol was significantly
                                                                             (P < 0.05) decreased by each plant sterol at all time
                                                                             points. Sitosterol inhibited cholesterol absorption by 57%
                                                                             at 24 hr compared to 41% inhibition with fucosterol. This
                                                                             difference was significant (P < 0.05). The absorption of
                                                                             sitosterol and fucosterol from the binary mixtures was less
                                                                             than 2 % in 24 hr. The average lymph flow was constant at
                   3      6      9                4                 24       each time in the three groups with a total volume (ml) in
                                                                             24 hr of 185 f 13, 163 17, and 151 19, respectively *
                                    Time (Hours)                             (data not shown).
Fig. 1. Inhibition of exogenous cholesterol absorption by sitosterol and
fucosterol. Lymph fistula rats were administered intragastricallyan emul-    Micellar cholesterol absorption in the presence of
sion containing 25 mg [3H]choiesterol(C) alone or plus 25 mg of either       micellar sitosterol or fucosterol
sitosterol (S) or fucosterol (F). Lymph was collected with time for 24 hr
and analyzed for sterol radioactivity (C) by liquid scintillation counting     In bile fistula rats, receiving “artificial”bile (no sterols),
or sterol mass (S and F) by GLC. Each data point is the mean SE for
n 6; ‘C group significantly different from C + S and C + F groups,
P < 0.05; ‘*C + S group significantly different from C + F group,
                                                                             the lymphatic absorption of cholesterol alone and in the
                                                                             presence of plant sterols was followed for 24 hr after intra-
P < 0.05.                                                                    duodenal administration of the micellar-solubilizedsterols.

1576       Journal of Lipid Research Volume 29, 1988
The results shown in Fig. 2 indicate that neither micellar               3.0
sitosterol nor fucosterol inhibited the lymphatic absorption                                       Sitosterol (S)
of micellar cholesterol. In addition, although in micellar
solution, sitosterol was poorly absorbed from the binary                 2.5
mixture with about 5% of the labeled dose appearing in
the lymph in 24 hr.
   In a second study, the “artificial”bile infusate contained             2.0
150 pM cholesterol ( 5 mg sterol delivered overnight) or
150 pM of both cholesterol and sitosterol ( 10 mg sterols                                      Fucorterol (R .i
                                                                                                              ‘     /
delivered) to increase the sterol challenge. As in the first              1.5
                                                                                                   ,    “,:p‘/
study, micellar sitosterol did not inhibit the absorption of                                                 I
                                                                                                             .   e c+s         (1:l)

micellar cholesterol (results not shown).

Micellar solubility of cholesterol, sitosterol, and                       1.o
fucosterol alone and in binary combinations                                                :                              Cholesterol (C)
in vitro                                                                                                                          .
   A set of “solubility” curves for individual and binary                 0.5

(equimolar) mixtures of sterols solubilized in micelles con-
taining 6.6 mM sodium taurocholate, 0.6 mM egg yolk
phospholipids, 1 mM oleic acid, and 0.5 mM monoolein is

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shown in Fig. 3. Sterol “solubility”was assessed qualita-                       0        0.4           0.8          1.2        1.6          2.0

tively by absorbance measurement at a wavelength of 430                                                 Total Sterols (mM)
nm. The initial preparations of individual sterols were clear      Fig. 3. Micellar solubility of sterols alone and in binaly mixtures.
with a baseline absorbance of 0.1. However, as the concen-         Cholesterol (C), sitosterol (S), and fucosterol(F) done and C + S,C +F,
tration of each sterol was increased, turbidity appeared,          and S + F in equimolar binary mixtures containing 0.2 to 2.0 mM totd
                                                                   sterol were sonicated at 37OC in 2.0 ml o 15 mM sodium phosphate
although at different concentrationsfor each, and the sterols      buffer, pH 7.4, containing 6.6 mM sodium taurocholate, 0.6 m egg yolk
precipitated upon standing. Specifically, the micellar solu-       phospholipids, 1 m oleic acid, 0.5 m monoolein, and 132 m sodium
                                                                                       M                   M                       M
tions containing either cholesterol, fucosterol, or sitosterol     chloride. Turbidity was measured spectrophotometridy at 430 nm. Sus-
                                                                   pending solution (no sterols) absorbance = 0.101, The results are the mean
were clear at sterol concentrations up to 0.8 mM, 0.6 mM,          for n = 2. Variation in absorbance 51.0%. ‘Turbidity or precipitateswere
and 0.27 mM, respectively. This suggests that cholesterol          observed.
is more soluble than fucosterol which is more soluble than
sitosterol. In binary mixtures prepared with cholesterol and
sitosterol (M), precipitate appeared at a total sterol con-
                 a                                                 sitosterol for a total sterol concentration of 0.55 mM, and
centration of 0.4 mM and absorbance rose sharply after             0.49 mM cholesterol and 0.35 mM fucosterol for a total
this. By contrast, equimolar combinations of fucosterol and        sterol concentration of 0.84 mM. These represent 66% and
either cholesterol or sitosterol up to 2 mM total sterol dis-      50% less micellar cholesterol, respectively, than that in bile
played no turbidity even after standing overnight.                 salt micelles prepared with 1 mM cholesterol alone. How-
   Since absorbance measurements are indicators of micel-          ever, with the cholesterol-fucosterol mixtures, the amount
lar size and provide only a “qualitative”estimate of sterol        of each sterol in the clear supernatant was dependent upon
solubility, the micellar solubility of cholesterol and how it is   the centrifugal force, with 1.7 mM total sterol determined
affected by plant sterols was assessed quantitatively by the       in the 10,000 g supernatant and half this amount in the
standard ultracentrifugation method. Micellar solutions            100,000 g supernatant. This anomalous behavior of the
prepared with either 1 mM or 2 mM [3H]cholesterolalone             cholesterol-fucosterol mixture was not observed with
or with 1 mM [3H]cholesterolplus either 1 mM [ ‘‘C]sitos-          cholesterol alone and to a very limited extent with the
terol or 1 mM fucosterol were incubated at 37OC overnight.         cholesterol-sitosterol mixture. The above results were un-
After centrifugation the micellar concentration of each            changed relatively (data not shown) when the sterols were
sterol was measured in the clear supernatants (micellar            solubilized in bile salt micelles containing either phos-
phase). As shown in Table 1, about 95% of the original             pholipids or monoolein and oleic acid.
cholesterol was in micellar solution after centrifugation at
100,000g. Although some degree of turbidity was visual-            Micellar solubility of cholesterol alone and in binary
ized, no precipitate was collected at either concentration.        combination with sitosterol and fucosterol in vivo
With the binary mixtures of cholesterol and either sitosterol         As shown in Table 2, the percentage of [3H]cholesterol
or fucosterol, 1 mM each, the respective 100,000 g super-          in the micellar phase prepared from the lumenal content
natants contained 0.33 mM cholesterol and 0.22 mM                  collected from the small intestine was reduced 33% by

                                                                      Z k e h et al.   Inhibition of cholerteml absorption                        1577
                         TABLE 1. Micellar solubility of cholesterol alone and in binary mixtures with plant sterols

                                                                                        M   Sterol in Supernatant at

                        Group                    Sterol          10,000 g              25,000 g             50,000 g              100,000 g

               Cholesterol (1 mM)                  C               0.99                 0.98                 0.96                  0.94
               Cholesterol (2 mM)                  C               1.9                  1.8                  1.8                   1.8
               Cholesterol (1 mM)    +             C               0.42                 0.40                 0.37                   0.33
                 Sitosterol (1 mM)                 S               0.29
                                                                   -                    0.27
                                                                                        -                    0.25
                                                                                                             -                     0.22
                   Sum                                             0.71                 0.67                 0.62                   0.55
               Cholesterol (1 mM) +                C               0.93                 0.82                 0.67                  0.49
                 Fucosterol (1 mM)                 F               0.75
                                                                   -                    0.64
                                                                                        -                    0.49
                                                                                                             -                     0.35
                   Sum                                             1.68                 1.46                  1.16                 0.84

                  [3H]Cholesterol alone (1 and 2 mM) or in binary mixtures containing [SH]cholesterol (1 mM) plus either
               ["C]sitosterol (1 mM) or fucosterol (1 mM) were sonicated for 2 min at 37OC in 12.0 ml of 15 mM sodium phos-
               phate buffer, pH 7.4, containing 6.6 mM sodium taurocholate, 0.6 mM egg yolk phospholipids, 1 mM oleic acid,
               0.5 m monoolein, and 132 mM sodium chloride. The solutions were divided into 3-ml aliquots, incubated at 37OC
               overnight, and centrifuged at the indicated force for 60 min at 37°C. The supernatantswere collected, and cholesterol
               (C) and sitosterol (S) concentrations were determined by liquid scintillation counting and fucosterol (F) concentra-
               tion was determined by gas-liquid chromatography. The results are the mean for n          2. Variation in mM sterol
               values 5 2 % .

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sitosterol and 25% by fucosterol. On the basis of the                       Competition of micellar sitosterol and fucosterol with
cholesterol content per ml of micellar phase, the concen-                   micellar cholesterol for binding to mucin
tration of cholesterol alone was estimated at 1.0 mM. This                    The mucin binding of cholesterol and plant sterols from
was reduced to 0.67 mM and 0.75 mM by sitosterol and                        micellar binary mixtures containing cholesterol (60 pM)
fucosterol, respectively.                                                   and either sitosterol (60 or 120 pM) or fucosterol (60 pM)
                                                                            was independent as shown in Fig. 6.
Concentration dependency of micellar sterol binding to
brush border membranes                                                      Competition of sitosterol with cholesterol
                                                                            for esterification
   Brush border membranes incubated with increasing
(25-325 pM) concentrations of either [3H]cholesterol or                       Cholestml esterase: Cytosolic cholesterol esterase in the
[14CC]sitosterol micellar solution bound both sterols. As
               in                                                           presence of saturating levels of sterol (0.5 pmol) esterified
shown in Fig. 4, two- to threefold more cholesterol than                    [3H]cholesterolat more than twice the rate of [4- '4C]sitos-
sitosterol bound at each concentration. Binding did not
reach a maximum at the highest level of cholesterol,
although at cholesterol concentrations greater than 100 p M ,                   TABLE 2 . Effect of sitosterol and fucosterol on the micellar
binding was no longer linear. In contrast, binding reached                             solubility of cholesterol in lumenal content
a maximum as 200 pM sitosterol was approached.                                                                               Micellar Cholesterol

                                                                                                                 Percentage of
Competition of micellar sitosterol and fucosterol with                          Group                             Dose Fed                          fig12 ml
micellar cholesterol for binding to brush
                                                                            Cholesterol                         3.22   f   0.29                 805    f   73
border membranes                                                            Cholesterol +
   The brush border binding of cholesterol and sitosterol                     sitosterol                        2.13 f 0.46                     532    *   115
                                                                            Cholesterol +
from micellar binary mixture was independent at low total                     fucosterol                        2.41 i 0.18                     602    +   45
sterol concentrations (I p M , 1C:2S) as shown in Fig. 5,
panel A. In contrast, in a second study (Fig. 5, panel B)                      Rats that were meal-fed a semi-purified diet (10 g) containing 0.25%
                                                                            cholesterol (C) alone or plus 0.25% sitosterol (C + S) or fucosterol
that included a fucosterol group and where the total sterol                 (C + F) for 7 days were killed 2 hr after they consumed the final meal
concentration in the micellar binary mixtures was increased                 to which 5 pCi of [SH]cholesterolwas added. Lumenal content was col-
(200 p ~ 1C:lS or lF), the brush border binding of
             ,                                                              lected (2-3 ml), heated to inactive lipolytic enzymes, and centrifuged at
                                                                            100,000 g for 60 min. Micellar [3H]cholesterolin the dear supernatant
cholesterol was inhibited an average of 38% and 35% by                      was determined by liquid scintillation counting. Results are the
sitosterol and fucosterol, respectively.                                    mean   *  SE, n = 5.

1578     Journal of Lipid Research Volume 29, 1988
             loo   -                                                         tein in the absence or presence of an equimolar amount of
                                                                             sitosterol. [ '4C]Sitosterol (84 nmol) was not esterified in the
                                                                             absence or presence of added exogenous cholesterol. These
             75    -                                                         results (not shown) are in agreement with those reported
                                                                             by others (13).

              50   -                                                                                DISCUSSION

                                                                                The present study supports numerous previous reports
              25   -                                                         that plant sterols inhibit cholesterol absorption (1-3) and
                                                                             extends these studies to include fucosterol and to assess the
                                                                             mechanism of the inhibitory effects. The anticipated inhi-
                                                                             bition of absorption occurred only when sitosterol or
                                                                             fucosterol was given with cholesterol (25 mg each sterol)
                                                                             as an intragastric emulsion (Fig. 1). Comparatively, sitos-
                                    Sterol CUM)                              terol was a significantly more effective inhibitor of ex-
                                                                             ogenous cholesterol absorption than fucosterol(57 vs. 41%).
Fig. 4 Concentration dependency of sterol binding to brush border
membranes. Brush border membranes (2.0 mg protein) were incubated            However, neither plant sterol (50 p M ) inhibited cholesterol
in a volume of 5.0 ml at 37% for 30 min with increasing concentrations       absorption when co-administered with cholesterol (50 p ~ )

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(25 to 325 PM) of (SH]cholesterolor ("C]sitosterol in micellar solution      in micellar solution. This was true whether the micellar
containing 6.6 mM sodium taurocholate and 0.6 mM egg yolk phos-
pholipids. Sterol bound was determined by liquid scintilation counting.      sterol meal was given to bile-fistula animals receiving an
Sterol binding at zero time (< 10 nmollmg protein) was subtracted from       overnight continuous infusion of sterol-free (Fig. 2) or
that at 30 min. The results represent one study.                             sterol-loaded "artificial" bile (results not shown).
                                                                                Differences in feeding protocols per se, i.e., intragastric
terol (25 vs. 10 nmol ester formed/per mg protein). Fur-                     emulsions of sterols to animals with normal bile flow versus
ther, the rate of [ 3H]cholesterol esterification was not al-                intraduodenal micellar solutions of sterols to bile-fistula
tered by the addition of an equimolar amount (0.5 pmol)                      animals, were unlikely to have accounted for the difference
of sitosterol (results not shown).                                           in effect on cholesterol absorption. Predictably, emulsified
   ACAT Microsomal ACAT catalyzed [ '4C]cholesterol (84                      sterol (25 mg cholesterol + 25 mg sitosterol or fucosterol)
nmol) esterification at a rate of 560 pmol/min per mg pro-                   would be solubilized physiologically in the small intestine

 A            40                                                                    B                  T                           Cholesterol
        C                                                       Sitosterol


        g     30

        2 lo.

                        c      c + s c+2s             s                                                 C       C+S      C+F         S
Fig. 5 Competition of micellar sitosterol and fucosterol with micellar cholesterol for binding to brush border membranes. Brush border membranes
(2.0 mg protein) were incubated in a volume of 5.0 ml for 30 min at 37°C with micellar solutions containing 6.6 m M sodium taurocholate, 0.6 mM
egg yolk phospholipids, and sterols singly or in binary mixtures as indicated. Panel A: C, 25 pM [sH]cholesterol; C + S, 25 p~ [SH]cholesterol+ 25
    [14C]sitosterol; + 2S, 25 pM [JH]cholesterol + 50 pM ["C]sitosterol; and S, 25 pM sitosterol. Panel B: C, 100 pM [3H]cholesterol;C + S, 1 0 0 ~ ~
[sH]cholesterol + 100p~ ["CJsitosterol; C + F 100 pM [sH]cholesterol + 100 p M fucosterol; and S, 100 pM ["C]sitosterol. Sterols bound were deter-
mined by liquid scintillation counting. Results are the mean f SE for n = 3 in both studies.

                                                                               Ikah et al.   Inhibition of cholesterol absorption         1579
                       0 Cholesterol                                         cholesterol, in that order. Equimolar binary mixtures of
                           Sitosterol                                        sterols ranging in total sterol concentration from 0.2-2 mM
                                                                              behaved anomalously by these same measurements. Speci-
                                                                              fically in the cholesterol + sitosterol combination, turbidity
                                                                             appeared and sterol precipitated at a lower total sterol con-
                                                                             centration than with cholesterol alone (0.4 mM total sterol
                                                                             vs. 0.8 mM cholesterol). However, equimolar binary com-
                                                                             binations of sitosterol + fucosterol and cholesterol + fucos-
                                                                             terol remained dear at total sterol concentrations of 2 mM.
                                                                             Curiously, fucosterol, which differs from sitosterol by one
                                                                             double bond in the side chain, appeared much less effec-
                                                                             tive than sitosterol in restricting the micellar solubility of
                                                                             cholesterol. When the quantitative effect of these two plant
                                                                             sterols on the micellar solubility of cholesterol was assessed
                                s    c+s                                     by the conventional ultracentrifugation approach (Table l),
                                                                             the 100,000g supernatants from micellar solutions prepared
Fig. 6.   Competition of micellar sitosterol and fucosterol with micellar
cholesterol for binding to mucin. Mucin (10 mg protein) was incubated
                                                                             with 1.0 mM each of cholesterol and sitosterol contained
in a volume of 4.0 ml for 15 min at 37OC with micellar solutions contain-    0.33 mM cholesterol while those prepared with 1 mM each
ing 6.6 mM sodium taurocholate, 0.6 mM egg yolk phospholipids, and           of cholesterol and fucosterol contained 0.49 mM cholesterol.
sterols singly or in mixtures as indicated: C, 60 pM [JH]cholesterol;
c + s, 60 pM [JH]cholesterol + 60 pM ["C]sitostero~ c + 2s. 60 pM            It was noted in this study, as in the spectrophotometric

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[JH]cholesterol + 120 p~ ['4C]sitosterol; C + F, 60 pM [3H]cholesterol       study, that the cholesterol + fucosterol combination gave
 + 60 pM fucosterol; and S , 60 pM ["]sitosterol. Sterols bound were de-     rise to clear solutions, but the micellar concentration of each
termined by liquid scintillation counting. Rwults are corrected for sterol
binding at zero time ( e 2 0 nmolhg protein). Results are the mean for
                                                                             sterol decreased sharply with increased centrifugal force. We
n = 2. Variation in nmol incorporationhg protein 5 5.                        have not attempted to characterize this effect, which may be
                                                                             explained by the generation of either unstable micelles or,
                                                                             possibly, a mixed population of micelles of variable density.
at a concentration of approximately 1 m M total sterol in                    The in vitro effects of sitosterol and fucosterol on the micel-
micelles formed from the biliary phospholipids and bile                      lar content of cholesterol hold up in every particular in vivo
salts. Dilution with endogenous cholesterol (well less than                  (Table 2). The magnitude of the in vivo effect with each
1.0 mg contributed during absorption since bile contains                     plant sterol was less, but normal bile contains chenodeox-
about 0.15 mg/ml per hr in a 200-g rat) would be insignifi-                  ycholate in which cholesterol can be solubilized and predic-
cant. By comparison, the small intestine of bile-fistula rats                tably (10) not displaced by plant sterols. Overall, since
which had continuously received "artificial" bile supple-                    solubilization of cholesterol in bile salt micelles is a require-
mented with micellar sterols (about 5 mg cholesterol + 5                     ment for its efficient absorption ( 2 5 , 26), the displacement
mg sitosterol or fucosterol delivered overnight) was presented               of cholesterol from micelles by plant sterols in general and
with 0.3 mM total sterol. Thus, the major difference be-                     the greater displacement with sitosterol relative to fucosterol
tween the two feeding protocols was that, in the former,                     measured in vitro and in vivo are consistent with the ob-
sterol selection for micellar solubilization was by physiologi-              served inhibitory effects on cholesterol absorption in vivo.
cal choice and in the latter it was not (both sterols were in                Moreover, these data are consistent with a recent report
micellar solution prior to infusion). As the result of this                  (10) in which the importance of the side chain substitution
selection process, the data suggest that plant sterols were                  at carbon 24 to sterol solubilization in taurocholate micelles
selected preferentially and limited the micellar solubilization              in vitro was demonstrated. Sitosterol, which is less hydro-
of cholesterol. This is borne out by the in vivo study in                    philic than cholesterol, has a lower capacity but higher
which the effect of sitosterol on the lumenal micellar                       affinity for binding to cholic acid micelles and was predicted
concentration of cholesterol was assessed (Table 2).                         to displace cholesterol with a favorable free energy change.
Mechanistically, the simplest explanation of the limitation                  Our recent in vitro studies (9) revealed that the rate of
would be sterol dilution, but this does not explain the                      sitosterol movement from the micellar phase to triolein was
significant difference in inhibitory effectiveness between                   3.5-fold less than cholesterol. This is consistent with the
sitosterol and fucosterol.                                                   suggested differences in their micellar affinity. By the same
    In vitro studies (Fig. 3, Table 1) were designed to further              reasoning, it is predictable that fucosterol, to a lesser ex-
characterize the in vivo inhibitory effects of plant sterols                 tent, would displace cholesterol from the micelle. Related
on cholesterol absorption. For the individual sterols, the                   to this, in feeding studies, sitosterol is reported to produce
 turbidity data pointed to the formation of larger, less sta-                a greater hypocholesterolemic effect than fucosterol when
 ble micelles with sitosterol followed by fucosterol and                     each is included at 1.0% in the diet of rats (27).

1580       Journal of Lipid Research Volume 29, 1988
   Although displacement of cholesterol from micelles may            as the sole substrate, was esterified at about half the rate
represent the major mechanism of plant sterol-mediated               of cholesterol as reported earlier (11) but failed to compete
inhibition of cholesterol absorption, other mechanisms have          with cholesterol when present at equimolar concentrations.
been suggested. These include plant sterol inhibition of             Since the esterifying enzymes either do not or poorly use
cholesterol binding to brush border membranes andor cell             sitosterol as a substrate and because sitosterol uptake into
surface mucins and inhibition of intracellular cholesterol           the cell is extremely limited, these enzymes cannot account
esterification.                                                      for plant sterol inhibition of cholesterol absorption.
    With respect to the cell surface, early studies (28) sug-           The basis for the inhibitory action of plant sterols on
gested the presence of specific binding sites for cholesterol.       cholesterol absorption has been assessed. Plant sterol inhi-
If such sites exist, micellar sitosterol did not compete with        bition of cholesterol absorption appears unrelated to com-
micellar cholesterol for these in rat jejunal loops in situ (8).     petition with cholesterol for binding to the cell surface mem-
In addition, during absorption no accumulation of sitosterol         brane or its mucin coat. Neither are the plant sterols
occurs in and/or on the intestine (e.g., refs. 9, 12). Consis-       competitive inhibitors of the cholesterol esterifying en-
tent with this, the present in vitro study confirmed indepen-        zymes. Rather, the data demonstrate that plant sterols res-
dence of binding of the two sterols to isolated brush border         trict the solubility of cholesterol in micelles prepared with
at low micellar sterol concentrations (Fig. 5A, 25 and 50            taurocholate, the principal bile salt in rat and human bile
pM) but at higher levels of cholesterol (100 pM) in combi-           (31). Sitosterol relative to fucosterol restricts the micellar
nation with either sitosterol or fucosterol (100 fiM), less          solubility of cholesterol more severely, which accounts for
 (-38%) cholesterol bound (Fig. 5B). At the higher total             its greater inhibitory effect on cholesterol absorption. M
 sterol concentration, this result is expected since sterol bind-

                                                                                                                                             Downloaded from by guest, on September 23, 2011
 ing to the brush border is approaching saturation (Fig. 4).         This research was supported in part by Public Health Services
    To the extent that these in vitro and the in vivo results        Grant HL 32982.
 are comparable, cholesterol absorption was not inhibited            Manurcripf received 21 scptcmbcr 1987 and in mbed fonn 18 May 1988.
 when intestine was exposed to a continuous overnight
 challenge with 150 pM each of cholesterol and sitosterol,
 a concentration of total sterol that was 100 pM greater than
 that which produced competition in vitro. This may be ex-
 plained by the greater accessibility of sterols to isolated          1. Subbiah, M. T. R. 1973. Dietary plant sterols: current sta-
 brush border which has a minimal unstirred water and mu-                tus in human and animal sterol metabolism. Am J. Clin. Nutr:
 cin layer and to differences in a closed in vitro versus an             26: 219-225.
 open in vivo system. However, under physiological condi-             2. Pollak, 0.J., and D. Kritchevsky. 1980. Sitosterol. In Mono-
                                                                         graphs on Atherosclerosis. T. B. Clarkson, D. Kritchevsky,
 tions, the results suggest that the monomolecular concen-
                                                                         and 0. Pollak, editors. Karger, Basel. 1-215.
 tration of sitosterol attained at the cell surface was below         3. Vahouny, G.V., and D. Kritchevsky. 1981. Plant and marine
 that required for competition for binding.                              sterols and cholesterol metabolism. In Nutritional Pharma-
    Mucins which blanket the cell surface bind cholesterol               cology. G. Spiller, editor. Alan R. Liss, New York. 32-72.
 (29) and may represent a barrier through which cholesterol            .
                                                                      4 Kuksis, A., and T. C. Huang. 1962. Differential absorption
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 must pass in the manner suggested for the unserred water
                                                                      5. Vahouny, G. V., W. E. Connor, S. Subramaniam, D. S. Lin,
 layer (30). If so,plant sterols might be competitive for bind-          and L. L. Gallo. 1983. Comparative lymphatic absorption
 ing. When sterols, singly and in mixtures, were incubated               of sitosterol, stigmasterol, and hcosterol, and differential in-
 with mucin, a small sterol binding differential of less than            hibition of cholesterol absorption. Am. J. Clin. Nutr. 37:
 2:l was observed in favor of cholesterol, and sterol binding            805-809.
                                                                      6. Sugano, M., H. Morioka, and I. Ikeda. 1977. A compari-
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                                                                         son of hypocholesterolemic activity of @-Hitosteroland @-
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 fucosterol). However, as mentioned above, concentrations             7. Child, P., and A. Kuksis. 1983. Uptake of 7-dehydro deriva-
 of plant sterols attained in the aqueous phase under phys-              tives of cholesterol, campesterol, and @-sitosterolby rat
 iological conditions are likely below that required for bind-           erythrocytes, jejunal villus cells, and brush border mem-
 ing competition with cholesterol.                                       branes. J. Lipid Res. 24: 552-565.
                                                                      8. Ikeda, I., and M. Sugano. 1983. Some aspects of mechanism
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1582       Journal of Lipid Research Volume 29, 1988

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