Lipids

Pectin Isolated From Prickly Pear (Opuntia sp.)

Modifies Low Density Lipoprotein Metabolism in
Cholesterol-Fed Guinea Pigs12
MARIA LZFERNANDEZ,3

AUGUSTO

TREJO* AND DONALD J. McNAMARA

Department of Nutrition and Food Science, and Nutritional Sciences Program, University of Arizona,
Tucson, AZ 85721 and *CIIDIR-IPN,
unidad Michoacan, Justo Sierra #28 Ote. Jiquilpan de Juarez,
Michoacan, Mexico 59510

INDEXINGKEYWORDS:
pectin
dietary cholesterol
low density lipoprotein
hepatic HMG-CoA redactase

guinea pigs
'These studies were supported in part by a grant-in-aid from the
American Heart Association, Arizona Affiliate (G-2-15-87), a grant
from the National Dairy Council, and funds provided by the Univer
sity of Arizona BiomdicalResearch Support Grant Program.
2Paper no. 7179 from the University of Arizona Agricultural Exper

Epidemiologie studies have associated consumption

of high fiber diets with a decreased risk for cardiovascu
lar disease (1-4). Dietary fiber has been reported to
reduce plasma cholesterol levels by binding to bile acids
and increasing bile acid fecal excretion (5). Because most
catabolism of cholesterol is by conversion to bile acids,
a dietary fiber-mediated
increase in the excretion of
neutral and acidic steroids will increase cholesterol
catabolism and lower plasma cholesterol levels. How

iment Station.
Postdoctoral fellow of the American Heart Association, Arizona
Affiliate.
""Abbreviations: apo, apolipoprotein; HC diet, high cholesterol diet;
HC-P diet, high cholesterol plus pectin diet; HDL, high density
lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl
coenzyme A;
LDL, low density lipoprotein; VLDL, very low density lipoprotein.

0022-3166/90 $3.00 1990 American Institute of Nutrition. Received 14 December 1989. Accepted 4 May 1990.

1283

Downloaded from jn.nutrition.org by guest on October 9, 2011

ever, the type and amount of dietary fiber and the

presence of other nutrients in the diet influence the
biochemical properties of fiber and its effect on endog
enous cholesterol and lipoprotein metabolism.
In studies of dietary fiber, intake of pectin, guar gum,
oat bran and other soluble fibers has consistently re
sulted in a decrease in plasma total and low density
lipoprotein (LDL)4 cholesterol levels in humans (6-12)
and animal models (13-16). In contrast, intake of insol
uble fibers such as cellulose and wheat bran has been
reported to have either a hypercholesterolemic
effect
(17, 18) or no effect on plasma cholesterol levels (13, 19)
in animal studies.
Using animal models, various studies have investi
gated the effect of dietary fiber on hepatic 3-hydroxy-3methylglutaryl
coenzyme A (HMG-CoA) reduc-ase
(20-22), hepatic cholesterol levels (17, 18), fecal bile acid
excretion (23, 24) and plasma apolipoprotein (apo) A-I
concentrations (25). However, the effects of dietary fiber
on LDL metabolism and LDL receptor levels have not
been addressed in detail.
The present study was undertaken to evaluate the
effects of pectin isolated from prickly pear (Opuntia sp. )
on several parameters of cholesterol and lipoprotein
metabolism in guinea pigs. Because this pectin has been

ABSTRACT The effect of prickly pear soluble fiber on

low density lipoprotein (LDL) metabolism was investi
gated by feeding male guinea pigs either a nonpurified
diet containing 0.25% cholesterol (HC diet) or the HC
diet + 1% prickly pear pectin (HC-P diet). Plasma
cholesterol levels were significantly decreased by the
HC-P diet, with a 33% decrease in LDL levels (p <
0.02) and an increase in LDLdensity. Hepatic free and
esterified cholesterol levels were reduced 40 and 85%,
respectively (p < 0.002), by the HC-P diet. Hepatic
microsomal 3-hydroxy-3-methylglutaryl coenzyme A
reduc-aselevels were not different. 125I-LDLbinding to
hepatic membranes was increased 1.7-fold by the
HC-P diet (p < 0.001), with receptor affinity (Kd)being
unaltered and receptor number (Bmax)being signifi
cantly increased (p < 0.001). These data suggest that
prickly pear pectin may act by a mechanism similar to
that of bile acid-binding resins in lowering plasma
cholesterol levels. The observed reduction in LDLand
hepatic cholesterol levels and increase in LDLdensity
and hepatic apolipoprotein B/E receptors are re
sponses suggesting an increased demand on hepatic
cholesterol from increased excretion of bile acids and
interruption of the enterohepatic circulation. J. liutr.
120:1283-1290, 1990.

1284

FERNANDEZ ET AL.

used successfully to treat diabetic patients at concentra

tions as low as 60 mg/d (A.Trejo, unpublished data), the
effects of this soluble fiber on plasma lipid levels, he
patic cholesterol content, hepatic HMG-CoA reduc-ase
and LDL binding to guinea pig hepatic membranes were
investigated.
Like humans, guinea pigs have high levels of LDL
relative to levels of high density lipoprotein (HDL).
They also have shown a reduction in plasma total and
LDL cholesterol levels in response to a polyunsaturated
fat diet (26, 27) and have shown an increase in plasma
total cholesterol when fed high levels of dietary choles
terol (28). For these reasons, the guinea pig was chosen
as the experimental animal model to measure the ef
fects of prickly pear pectin on cholesterol and lipopro
tein metabolism.
MATERIALS AND METHODS

was based on density fractions: d < 1.019g/mL for VLDL;

d 1.019-1.090 for LDL; and d 1.090-1.21 for HDL. He
patic concentrations of total and free cholesterol were
measured according to the method of Sale et al. (31).
Cholesterol ester concentration was determined by sub
tracting free from total cholesterol.
11)1 isolation and characterization. Plasma was ob
tained from pooled blood samples of guinea pigs fed both
diets. Previous studies have demonstrated that the
source of LDL does not affect apo B/E receptor affinity
(Kd)for the ligand (27).Plasma lipoproteins were isolated
by adjusting the plasma density to 1.25 g/mL with solid
KBr and by centrifuging for 36 h at 125,000 x g at 15C
in a Beckman Ti-50 rotor. The isolated lipoprotein frac
tion was adjusted to a density of 1.3 g/mL with KBr,and
a 10-mL volume was overlayered with 30 mL of 0.9%
NaCl solution in a Quick Seal centrifugation tube (Beck
man Instruments, Palo Alto, CA). Centrifugation was
performed in a VC-53 vertical rotor (Beckman Instruments)for3hat 100,000xgat 10C
to generate a density
gradient fractionation of the lipoproteins (32).The lipo
protein profile was determined by measuring choles
terol in the isolated fractions, and density values were
determined by measurement of the refractive index (33).
The fractions corresponding to LDL were pooled from
the d 1.02-1.09 g/mL portion of the gradient. To confirm
the purity of LDL in the pooled fractions, sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
mini gels (4.5 to 20% linear gradient) were run for 45
min at a constant voltage, 18-20 mA, at room tempera
ture and stained with Coomassie blue (34).
Microsome isolation and hepatic HMG-CoA reduc
-aseassay. Guinea pigs were killed at the nadir of the
diurnal rhythm, livers were removed and hepatic microsomes were isolated by pressing liver tissue through a
tissue grinder into 1:3 homogenization buffer (50
mmol/L KH2PO4,0.1 mol/L sucrose, 50 mmol/L KC1,30
mmol/L EDTA, and 2 mmol/L DTT, pH 7.2)containing
50 mmol/L NaCl (35). This crude preparation was fur
ther homogenized with a Potter-Elvehjem homogenizer.
A microsomal fraction was isolated by two 15-min
centrifugations at 10,000 x g (JA-20rotor, J2-21 centri
fuge, Beckman Instruments, Palo Alto, CA) followed by
a 1-h centrifugation at 100,000x g in a Ti-50 rotor at 4C.
Microsomes were resuspended in the homogenization
buffer and washed for an additional hour at 100,000 x g.
After centrifugation, microsomal pellets were homoge
nized in a small volume of buffer and stored at -70C
(36). Microsomal HMG-CoA reduc-ase(EC 1.1.1.34)
activity was measured by the radioisotopic method of
Shapiro et al. (37).
Hepatic membrane isolation. Livers were homoge
nized with two 10-s pulses of a Polytron (Brinkmann
Instruments, Westbury, NY) at a setting of 10 in 10 mL

Downloaded from jn.nutrition.org by guest on October 9, 2011

Materials. DL-Hydroxy-[3-'4C]methylglutaryl coenzyme A (51.9 mCi/nmol) and DL-[5-3H]mevalonicacid

(48.6 mCi/nmol) were purchased from New England
Nuclear, Boston, MA. A cholesterol enzymatic kit, cho
lesterol oxidase, cholesterol esterase and horseradish
peroxidase were from Boehringer Mannheim, Indianap
olis, IN. DL-3-Hydroxy-3-methylglutaryl coenzyme A,
mevalonolactone, glucose-6-phosphate, glucose-6phosphate dehydrogenase and NADP were from Sigma
Chemical, St. Louis, MO. Halothane was obtained from
Halocarbon Laboratories, Hackensack, NJ.
Diets. Guinea pig nonpurified diet was obtained
from Teklad (Madison, WI). The diet is reported to be
17.9% protein (mainly soybean meal), 1.9% vegetable
fat, 49.3% carbohydrates and 14% fiber (mainly alfalfa);
minerals and vitamins, including vitamin C, are added
according to guinea pig requirements. Two diets were
formulated from a single batch of the nonpurified diet
by adding 0.25% (wt/wt) recrystallized cholesterol (HC
diet) or 0.25% recrystallized cholesterol +1% (wt/wt)
prickly pear pectin (HC-P diet). The diets were repelleted. Pectin was isolated from Opuntia sp. grown in
Jiquilpan Michoacan in Mexico (patent pending).
Animals. Male Hartley guinea pigs (Har-an
SpragueDawley, Indianapolis, IN) weighing between 250 and
300 g were randomly assigned to either the HC diet or
the HC-P diet. After 25 d of dietary treatment, the
animals were anesthetized with halothane vapors and
exsanguinated by cardiac puncture. Plasma was sepa
rated from blood cells for analysis of plasma lipids and
isolation of plasma LDL. Livers were removed for prep
aration of hepatic membranes and hepatic microsomes
and for determination of hepatic cholesterol content.
Plasma and liver lipids. Total plasma cholesterol
and triglycridelevels were determined by enzymatic
analysis (29). HDL cholesterol was determined by the
precipitation method of Warrick et al. (30). Very low
density lipoprotein (VLDL),LDL and HDL were sepa

rated by sequential ultracentrifugation in a L8-M ultracentrifuge (Beckman Instruments, Palo Alto, CA) at
125,000 x g at 15C
for 19 h in a Ti-50 rotor. Separation

1285

PECTIN AND LDL METABOLISM

of buffer A (150 mmol/L NaCl, 1 mmol/L CaCl2/ 10

mmol/L Tris-HCl at pH 7.5). Hepatic membranes were
isolated by ultracentrifugation
of an 8000 x g superna
tant (JA-20 rotor, J2-21 centrifuge) at 100,000 x g at 4C

100

in the presence of 1 mg/mL excess of either unlabeled

guinea pig or human LDL to determine receptor-medi
ated binding. After incubation, the membranes were
pelleted by ultracentrifugation
by overlayering 75 |iL of
the incubation mixture with 100 uL of newborn bovine
serum in Beckman cellulose propionate tubes and centrifuging at 100,000 x g for 45 min in a Ti 42.2 rotor (38).
After the supernatant was aspirated, the membranes
were washed with 125 uL of serum and centrifuged at
the same speed for 25 min. The supernatant was re
moved, the tubes sliced and the pellets counted in a
gamma counter (LKB-Wallac CliniGamma, Finland) for
radioactivity.
Previous studies have demonstrated
that human
HDL effectively competes with guinea pig 125I-HDLin
binding to guinea pig hepatic membranes at 37C(42).
To test whether human LDL could be used as an unla
beled competitor, competition studies were conducted
at 4 and 37Cusing human and guinea pig LDL as the
unlabeled competitors for hepatic binding of guinea pig
125I-LDL.Human LDL was a weak competitor for guinea
pig LDL at 4C;however, at 37Cand at high concentra
tions, unlabeled human LDL competed adequately with
labeled guinea pig LDL in binding to guinea pig hepatic
membranes (Fig. 1). On the basis of these results, human
LDL was used as the unlabeled competitor for all bind
ing assays at a concentration of 1 mg/mL.
Equilibrium parameters.
To determine
hepatic
membrane receptor affinity for LDL (Kd)and total apo
B/E receptor number (Bmax),isolated guinea pig LDL was
radioiodinated
and incubated at 37Cwith isolated
membranes in the presence of 1 mg/mL of unlabeled
human LDL over a range of 5 to 30 ug of LDL protein/mL. Scatchard analysis (43) was used to determine
Kd (ug LDL protein/mL) and Bmax(ng LDL bound/mg
membrane protein).

25

50

75

100

125

150

175

LDL FOLD EXCESS

FIGURE 1 Competition curves for LDL binding to guinea

pig hepatic membranes measured at 4 and 37C.Competition
with guinea pig (O) and human LDL at 4C(top) and 37C
(bottom). Studies were conducted using hepatic membranes
from guinea pigs fed the HC diet. Each point represents a single
determination. Guinea pig '"I-LDL was used at a concentra
tion of 5 ug/mL.

Statistical analysis. One-way analysis of variance

was used to assess differences in binding, in the equilib
rium parameters of Kd and Bmax,in cholesterol content
of livers and in plasma total, VLDL, LDL, and HDL
cholesterol levels. The least significant difference test
(44) was used to evaluate differences between means.
Data are presented as means so for the number of
assays shown.

RESULTS

Pectin effects on plasma and hepatic lipid levels.

Guinea pigs fed the HC-P diet exhibited a significant

26% reduction in plasma total cholesterol levels as
compared to levels in animals fed the HC diet (Table 1).
Triglycridelevels did not differ between animals fed
the two diets (Table 1).
As indicated in Table 1, a significant 33% decrease in
plasma LDL and HDL cholesterol levels was observed
in HC-P-fed guinea pigs. VLDL cholesterol concentra
tions were elevated in guinea pigs fed the HC-P diet,
although not significantly.
Fractionation of plasma lipoproteins by density gra-

Downloaded from jn.nutrition.org by guest on October 9, 2011

for 1 h. The isolated membranes were resuspended in

buffer A, flushed 10 times through a 22-gauge needle and
washed by centrifugation for 1 h at 100,000 x g and
stored in liquid nitrogen for use in binding assays (38).
For determination of LDL binding, frozen membranes
were thawed and resuspended in buffer B (100 mmol/L
NaCl, 0.5 mmol/L CaCl2, 50 mmol/L Tris-HCl, 20
mg/mL bovine serum albumin at pH 7.5) and flushed 10
times through a 22-gauge needle (39). Membrane protein
concentrations
were determined by the method of
Markwell et al. (40).
Binding assays. Pooled samples of guinea pig LDL
were radioiodinated
by the iodine monochloride
method as modified by Goldstein et al. (41) to give a
specific activity between 200 and 850 cpm/ng. Hepatic
membranes (200 ug protein) from guinea pigs were in
cubated with 10 ug/mL 125I-LDLprotein for 2 h at 37C

1286

FERNANDEZ ET AL.

HC.....0

TABLE 1

io-B

Plasma ipidsin guinea pigs fed nonpurified diet + 0.25%

cholesterol (HC diet) or nonpurified diet + 0.25% cholesterol
+ 1% pectin (HC-P diet)1

0>

E
o

Diet
Plasma lipids

CE

Ai

-
A\
*i
\i
\
e'42nf**,o \O'
i//
'
o
*-

HC-P

HC

89 24'
54 14

"-o-oi5*0
'

O
"*
O~- 0 O-o

0-00

.- -
-
-.. ... -.. -.. 9m

66 29b
52 23

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

DENSITY (g/ml)

Lipoprotein cholesterol'
VLDL cholesterol
LDL cholesterol
HDL cholesterol

\,

U^

mg/100 ml
Total cholesterol2
Total triglycrides2

HC_pli?

5 2
59 24
22 T

10 12
39 15b
15 5b

'Data are presented as means so. Values in the same row with

dient vertical rotor ultracentrifugation

indicated the
presence of LDL subfractions in both groups (Fig. 2). In
the HC-diet animals, the LDL peak density was 1.040
g/mL. Adding pectin to the diet increased the LDL peak
density to 1.055 g/mL (Fig. 2).
Hepatic concentrations
of total, free and esterified
cholesterol were significantly reduced in guinea pigs fed
the HC-P diet (p < 0.002) (Table 2). However, total
hepatic HMG-CoA reduc-ase activity was not affected
by adding pectin to the HC diet (Table 2).
Pectin effects on LDL binding and equilibrium pa
rameters. Receptor-mediated binding of guinea pig 125ILDL to guinea pig hepatic membranes was significantly
increased in animals fed the HC-P diet. At a concentra-

tion of 10 u/mL, I25I-LDLbinding values were 459.8

91.0 (n - 10) vs. 792.7 225.2 (n = 11) ng bound/mg
membrane protein for guinea pigs fed the HC and the
HC-P diets, respectively (p < 0.001). A significant nega
tive correlation (r = -0.597, p < 0.005 was found between
plasma LDL cholesterol levels and receptor-mediated
LDL binding to guinea pig hepatic membranes for ani
mals fed the HC and HC-P diets (Fig. 3).
A typical saturation curve for the binding of LDL to
guinea pig hepatic membranes for animals fed the HC-P
diet is shown in Figure 4. Total binding increased with
increasing concentrations of 125I-LDL,nonspecific bind
ing exhibited linearity and specific binding reached sat
uration at a concentration of 20 ug/mL.
Concentration-dependent
binding curves for the
binding of LDL to guinea pig hepatic membranes from
both groups of animals are shown in Figure 5. Both
membrane preparations
exhibited saturation at 20
ug/mL. Scatchard plots are shown in the inset of Figure

TABLE 2

Hepatic HMG-CoA reduc-ase levels and cholesterol content in guinea pigs fed nonpurified diet + 0.25% cholesterol {HC diet)
or nonpurified diet + 0.25% cholesterol + 1% pectin (HC-P diet)1

Reduc-ase3pmol/(min-mg

DietHC

protein)2.03

HC-PTotal6.1

1.3*
1.9"
2.6 0.8"Cholesterol2Freemg/g4.1
2.3 0.6"Ester2.0

0.9a
0.3 0.3bHMG-CoA

0.32
1.99 0.31

'Data are presented as means so. Values in the same column with different superscripts are significantly different (p < 0.002) as assessed by
ANOVA and the least significant difference test.
2n 10 for guinea pigs fed the HC diet, and n = 11 for animals fed the HC-P diet.
3n =
5 for each diet group.

Downloaded from jn.nutrition.org by guest on October 9, 2011

different superscripts are significantly different (p < 0.02) as assessed

by ANOVA and the least significant difference test.
2n = 22 for guinea pigs fed the HC diet, and n = 11 for animals fed
the HC-P diet.
3n - 10 for guinea pigs fed the HC diet, and n = 11 for animals fed
the HC-P diet.

FIGURE 2 Typical density fractionation profiles of plasma

lipoproteins of guinea pigs fed either the HC (O) or HC-P ()
diet. Plasma lipoproteins were fractionated by density gradi
ent ultracentrifugation
in a VC-53 rotor as described by
Poumay and Ronveaux-Duval (32).

1287

PECTIN AND LDL METABOLISM

O HC Dlat
HC-P Diet

1200O)

E
en

900

Q.
O>

O)
C

600

TD
C

m
_i
Q

300

O
O

10

20

30

40

SO

60

70

LDL Cholesterol (mg/100

80

90

100 110

mL)

FIGURE 3 Regression analysis of relationship between

plasma LDL cholesterol levels and receptor-mediated
LDL
binding in animals fed HC (O) or HC-P ()diets; r = -0.597 (p
< 0.005).

DISCUSSION
Effect of prickly pear pectin on plasma cholesterol
levels and lipoprotein profile. Pectin from various

2500

10

20

LDL CONCENTRATION

30
(M9/ml)

FIGURE 4 Saturation kinetics of the binding of guinea pig

LDL to a hepatic membrane preparation from an animal fed
the HC-P diet. Total binding (O) and nonspecific binding ()
were determined at 37Cin the absence and presence of 1
mg/mL unlabeled human LDL. Receptor-mediated binding (A)
exhibited saturation at a concentration of 20 \ig protein/mL.

250-

10

20

30

LDL CONCENTRATION
FIGURE 5 Binding kinetics and Scatchard plots (inset) for
receptor-mediated
binding (at 37C)of guinea pig LDL to
hepatic membranes from animals fed the HC ()or HC-P (O)
diets. In the Scatchard plot (inset), B represents bound ligand
(ng/mg) and B/F equals bound divided by free ligand
(ng/mg)/(ng/mL)].Ka values were 18.5 and 16.1 mg/mL, and
Bmaxvalues were 1300 and 2137 ng/mg protein for the animals
fed the HC and HC-P diets, respectively.

sources, including citrus pectin (45, 46) and other com

mercial brands (16, 47), has been consistently found to
reduce plasma cholesterol levels in animal models
when fed at levels ranging from 5 to 20% (13, 15,45,46,
48, 49). This cholesterol-lowering effect has been ob
served even in the presence of high fat (50) and high
cholesterol diets (47, 51). In clinical studies, pectin has
also shown a hypocholesterolemic effect in normal and
hyperlipidemic subjects (52, 53).
Similarly, we found that prickly pear pectin had a
hypocholesterolemic effect that affected both LDL and
HDL fractions when fed at a concentration as low as 1%.
Ney et al. (16) reported a normalization of rat plasma
lipoproteins from inclusion of oat bran in a hypercholesterolemic atherogenic diet; the major effect was in
the VLDL fraction. A decrease in the LDL and HDL
cholesterol was also reported, though apo A-I produc
tion was not affected. An increase in both LDL and HDL
plasma cholesterol levels has been documented in
guinea pigs fed a 0.25% cholesterol diet when compared
to animals fed nonpurified diets (28). In the present
study, adding pectin to the HC diet had a hypo
cholesterolemic effect in guinea pigs that resulted in
normalization of plasma LDL and HDL cholesterol
levels.
The hypocholesterolemic effect of pectin was not
consistent in all guinea pigs: some of the animals fed the
HC-P diet had normal plasma cholesterol values of

Downloaded from jn.nutrition.org by guest on October 9, 2011

5. A 1.5-fold increase in the apo B/E receptor Bmaxvalue

was observed for hepatic membranes from guinea pigs
fed the HC-P diet as compared to animals fed the HC
diet (2137 vs. 1300 ng/mg, respectively) whereas the
affinity constants for the apo B/E receptor for LDL were
the same for both membrane preparations (18.5 vs. 16.1
ug/mL) (Fig. 5). The equilibrium parameters of K<jand
Bmaxwere analyzed in four different membrane prepara
tions of both dietary groups. The affinity constant (Ka)
was not affected by diet, but the number of receptors
(Bmax)was significantly increased 1.5-fold for hepatic
membranes from guinea pigs fed the HC-P diet (p <
0.001 ((Table 3).

m
m
_i
o

1288

FERNANDEZ ET AL.

TABLE 3

Equilibrium constants for LDL binding to guinea pig hepatic

membranes from animals fed nonpurified diet + 0.25% cholesterol
(HC diet) or nonpurified diet + 0.25% cholesterol
+ 1% pectin (HC-P diet)1
Diet2

HC
HC-P

Bmax

\ig/mL

ng/mg protein

19.8 7.3
17.5 4.2

1628 316'
2385 342b

'Data are presented as means so. Values with different super

scripts in the same column are signfiicantly different (p < 0.02) as
assessed by ANOVA and the least significant difference test.
2n = 4 hepatic membrane preparations per group.

found a significanl reduction in hepatic cholesterol syn

thesis in animals fed the cellulose diet. In this study,
adding pectin did not modify the levels of hepatic HMGCoA reduc-ase activity that were almost completely
suppressed by the 0.25 % cholesterol diet.
Many investigalors have reponed a decrease in liver
cholesterol content from the addilion of peclin lo diels
(45, 48, 58). In ihis report a similar response was ob
served in lhal prickly pear peclin significanlly reduced
hepalic total, free and esterified choleslerol levels when
compared lo levels wilh ihe HC diel (p < 0.001) (Table
2). Like peclin, choleslyramine added lo a high choles
lerol diel also reduced hepalic choleslerol in rals (59).
Effect of pectin on LDL binding to hepatic mem
branes. The significanl increase in LDL binding ob
served for hepalic membranes from animals fed ihe
HC-P diel suggesls lhal iwo possible mechanisms may
be involved. One is an increase of receplor affinily for
ihe LDL particle, and ihe second is an increase in apo
B/E receplor number. Scalchard analysis demonslraled
lhal ihe Kd values were ihe same for bolh membrane
preparalions whereas Bmaxwas 1.5-fold higher for guinea
pigs fed ihe HC-P diel, indicaling lhal ihe number of apo
B/E recepiors was significanlly increased.

Comparative effects of cholestyramine and prickly

pear pectin on cholesterol metabolism. Peclin and the

Downloaded from jn.nutrition.org by guest on October 9, 2011

30-40 mg/100 mL reported for guinea pigs fed a nonpurified diet whereas others had values of 50-70 mg/100
mL, which are typical of guinea pigs fed the HC diet. It
has been found in this and other studies that the plasma
cholesterol levels for guinea pigs fed a high cholesterol
diet range from 50 to 140 mg/100 mL (28,54), suggesting
that some guinea pigs are hyper-responders to the HC
diet and others are not. A similar effect was observed
with the addition of pectin to the HC diet, in that some
guinea pigs had a more pronounced response than others
to prickly pear pectin in the presence of the high choles
terol diet. On average, an overall 26% reduction in total
cholesterol and 33% reduction in LDL and HDL choles
terol were observed (n = 11). Regression analysis of the
relationship between plasma LDL cholesterol levels and
LDL receptor binding indicated that there was a signif
icant negative correlation (p < 0.005) between these two
parameters. The observed correlation is consistent with
the hypothesis that apo B/E receptor expression is a
major determinant of plasma LDL cholesterol levels
(41). Because pectin was added to the HC diet at a
concentration
of only 1%, it would be of interest to
determine the hypocholesterolemic
response to prickly
pear pectin when fed at higher concentrations of 5-15%,
as used for studies of other fibers by various investiga
tors.
Prickly pear pectin effects on LDL density. As pre
viously reported for guinea pigs fed different dietary fats
(27), plasma from both groups of animals exhibited LDL
heterogeneity
characterized by the presence of LDL
subfractions (Fig. 2). It has been observed that LDL
density decreases to lower values when guinea pigs are
fed high cholesterol diets when compared to animals fed
nonpurified diets with low concentrations
of dietary
cholesterol (55). Adding pectin to the HC diet shifted
the LDL peak density from 1.040 to 1.055 g/mL (Fig. 2),
approaching values reported for guinea pigs fed nonpurified diets and heading toward the values of 1.071.08 g/mL reported for animals fed 7.5% dietary fat (26,
27).

These data suggest that consumption of pectin de

creases the size and increases the density of LDL. An
increase in LDL peak density also results from con
sumption of polyunsaturated versus saturated fat diets
(26, 27), and a decrease in LDL particle size results from
the intake of cholestyramine (56). Because larger, less
dense LDL particles contain more cholesteryl ester (27,
56), it would be predicted that pectin reduces cholesterylryl
ester concentrations of plasma LDL.
Pectin effects on hepatic cholesterol synthesis an
aid
cholesterol content. Adding 0.25% cholesterol to the
diet reduced guinea pig hepatic cholesterogenesis
by
90%. Values for hepatic HMG-CoA reduc-ase activity
for animals fed a nonpurified diet are 30-40 pmol/(min
mg protein) (N. Y. Yount and D. J. McNamara, unpub
lished data). Adding pectin to the cholesterol diet did
not affect hepatic cholesterol synthesis since both
groups of animals had similar levels of HMG-CoA reductase activity. Reports concerning the effects of oat
bran and pectin on hepatic HMG-CoA reduc-ase are
contradictory. Inclusion of oat bran decreased HMGCoA reduc-ase levels when compared to levels with a
semipurified control diet (22), and no effect on hepatic
HMG-CoA reduc-asewas observed by addition of cellu
lose or pectin to a semisynthetic diet (21). However,
Reiser et al. (20) reported an increased HMG-CoA reduc
-aseactivily in rals when peclin was added lo a semipurified diet. Illman et al. (57) compared the effects of
oat bran and cellulose on hepalic cholesterol synthesis
in rats; synlhesis was measured by incorporalion of
[3H]water inlo digilonin-precipilable
sierols. They

PECTIN AND LDL METABOLISM

7.

8.

9.

10.

11.

12.

13.

14.

15.
16.

17.

18.

19.
20.

21.

LITERATURE CITED
22.
1. TROWELL,H. (1975) Coronary heart disease and dietary fiber.
Am. J. Clin. Nutr. 28: 798-799.
2. KROMHOUT,D., BOSSCHIETER,
E. B. & DE LEZENNECOULANDER,
C.
(1982) Dietary fibre and 10-year mortality from coronary heart
disease, cancer and all causes. Lancet 1: 518-522.
3. KHAW,K. T. & BARRETT-CONNOR,
E. (1987) Dietary fiber and
reduced ischemie heart disease mortality rates in men and
women: a 12-year prospective study. Am. f. Epidemial. 126:
1093-1102.
4. ANDERSON,J. W. (1987) Dietary fiber, lipids and atherosclerosis.
Am. J. Caidiol. 60: 17G-22G.
5. KRITCHEVSKY,
D. (1978) Fiber, lipids and atherosclerosis. Am. J.
Clin. Nutr. 31:S65-S74.
6. IENKINS,D. J. A., REYNOLDS,
D., SLAVIN,B., LEEDS,A. R., JENKINS,

23.

24.

25.

A. L. &. JEPSON,E. W. (1980) Dietary fiber and blood lipids:

treatment of hypercholesterolemia with guar crispbread. Am. ].
Clin. ut-.33:575-581.
KiRBY,R. W., ANDERSON,J. W., SIEUNG,B., REES,E. D., LIN CHEN,
W. J., MILLER,R. E. &. KAY,R. M. (1981) Oat bran intake selec
tively lowers serum low-density lipoprotein cholesterol concen
trations of hypercholesterolemic
men. Am. f. Clin. Nutr. 34:
824-829.
SMITH,U. &. HOLM, G. (1982) Effect of a modified guar gum
preparation on glucose and lipid levels in diabetics and healthy
volunteers. Atherosclerosis 45: 1-10.
BEHALL,
K. M., LEE,K. H. & MOSER,P. B. (1984] Blood lipids and
lipoproteins in adult men fed four refined fibers. Am. J. Clin. Nutr.
39: 209-214.
ARO,A., UUSITUYPA,
M., VOUTILAINEN,
E. & KORHONEN,
T. (1984)
Effects of guar gum in male subjects with hypercholesterolemia.
Am. J. Clin. Nutr. 39: 911-916.
ANDERSON,J. W., STORY,L., SIEUNG,BL, LIN CHEN,W. ]., PETRO,
M.S.& STORY}. (1984) Hypocholesterolemic effects of oat-bran
or bean intake for hypercholesterolemic men. Am. J. Clin. Nutr.
40: 1146-1155.
SUPERKO,
R. H., HASKELL,
W. L., SAWREY-KUBICEK,
L. & FARQUHAR,
J. W. (1988) Effects of solid and liquid guar gum on plasma
cholesterol and triglycrideconcentrations in moderate hyper
cholesterolemia. Am. ]. Cardio!. 62: 51-55.
REDDY,B., WATANABE,
K. & SHEINHL,A. (1980) Effect of dietary
wheat bran, alfalfa, pectin and carrageenan on plasma cholesterol
and fecal bile acid and neutral sterol excretion in rats. /. Nutr. 110:
1247-1254.
GEE, J. M., BLACKBURN,
N. A. & JOHNSON,I. T. (1983) The
influence of guar gum on intestinal cholesterol transport in the
rat. Br. f. Nutr. 50: 215-224.
JUDD,P. A. &TRUSWELL,
A. S. (1985) The hypocholesterolaemic
effects of pectins in rats. Br. .Nutr. 53: 409-425.
NEY,D. M., LASEKAN,
J. B. & SHINNICK,F. L. (1988) Soluble fiber
tends to normalize lipoprotein composition in cholesterol-fed
rats. /. Nutr. 118: 1455-1462.
VAN BERESTEYN,
E. C. H., VAN SCHAIK,M. & MOGOT,M. F. K.
(1979) Effect of bran and cellulose on lipid metabolism in obese
female Zucker rats. /. Nutr. 109: 2085-2097.
PARK,C. S. & HARROLD,R. L. (1983) Effects of dietary protein
and fiber on growth and selected cholesterol responses of rats.
Ann. Nutr. Metab. 27: 137-144.
KIM, D. N., LEE,K. T, REINER,J. M. & THOMEAS,W. A. (1981)
Lack of beneficial effects of wheat bran cereals on cholesterol
balance in swine. Exp. Mol Pathol. 35: 301-313.
REISER,R., HENDERSON,
G. R., O'BRIEN,B. C. & THOMAS,J. (1977)
Hepatic 3-hydroxy-3-methylglutaryl
coenzyme A reductase of
rats fed semipurified and stock diets. /. Nutr. 107: 453-457.
PROIA,A. D., MCNAMARA,D. f., EDWARDS,
D. G. & ANDERSON,K.
E. (1981 ) Effects of dietary pectin and cellulose on hepatic and
intestinal mixed-function oxidations and hepatic 3-hydroxy-3methylglutaryl-coenzyme A reductase in the rat. Biochem. Pharmacol. 30: 2553-2558.
KELLEY,
f. J. & STORY,J. A. (1987) Short-term changes in hepatic
HMG-CoA reductase in rats fed diets containing cholesterol or
oat bran. Lipids 22: 1057-1059.
LAFONT,H., LAIRON,D., VIGNE,J. L., CHANUSSOT,
F., CHABERT,
C.,
PORTUGAL,
H., PAULI,A. M., CROTTE,C. & HAUTON,J. C. (1985)
Effects of wheat bran, pectin, and cellulose on the secretion of bile
lipids in rats. /. Nutr. 115: 849-855.
MATHE,D. & CHEVALIER,
F. (1981) Effects of dietary cholesterol,
L thyroxine, and various diets on the pools and fecal elimination
of bile acids in the rats. Nutr. Rep. Int. 23: 689-695.
HEIN, R. J., SCHOUTEN,J. A., VAN GENT, C. M., HAVEKES,
L. M.,
KOOPMAN,R. A. R. & VAN DERVEEN,E. A. (1984) the effect of
dietary linoleic acid and pectin on lipoprotein and apolipoprotein
AI concentrations in rhesus monkeys. Ann. Nutr. Metab. 28:
201-206.

Downloaded from jn.nutrition.org by guest on October 9, 2011

bile acid squestrant resin cholestyramine

appear to
have similar mechanisms in reducing plasma choles
terol levels. Cholestyramine
binds bile acids in the
intestine and interrupts their enterohepatic circulation.
As a result of this interruption,
three enzymes are
induced: phosphatidic acid phosphatase, cholesterol 7cchydroxylase and HMG-CoA reductase (60). Conse
quently, an increase in triglycrides, an increased
demand for intracellular cholesterol and an increased
cholesterol synthesis would be expected.
Similar to what has been reported for cholestyramine
(60, 61), prickly pear pectin may also affect cholesterol
absorption, as has been documented for citrus pectin
(62), and may increase bile acid fecal excretion, as has
been reported for some commercial pectin sources in
clinical (52) and animal studies (13).
Cholestyramine
has been shown to reduce plasma
total and LDL cholesterol levels (63, 64), decrease cho
lesterol hepatic content (59), decrease LDL particle size
and increase LDL peak density (56); increase receptormediated plasma clearance in rabbits (63) and humans
(64); and increase LDL binding to guinea pig hepatic
membranes (56). These observations are similar to what
has been reported in this study using prickly pear pectin.
Contrary to what has been reported for rats fed a high
cholesterol
diet treated with cholestyramine
(59),
prickly pear pectin did not increase hepatic HMG-CoA
reductase levels.
In summary, adding 1% prickly pear pectin to a high
cholesterol diet decreased total, LDL and HDL choles
terol levels (p < 0.02); altered LDL particle size as dem
onstrated by an increase in LDL peak density; decreased
hepatic concentrations of total, free and esterified cho
lesterol (p < 0.002); did not change hepatic HMG-CoA
reductase activity; and significantly increased the num
ber of hepatic apo B/E receptors (p < 0.001) without
modifying the receptor affinity. From these observa
tions, we conclude that the mechanisms involved in the
plasma hypocholesterolemic
effect of prickly pear pec
tin could be analogous to those reported for choles
tyramine and other bile acid-binding resins in clinical
and animal studies (59, 63, 64).

1289

1290

FERNANDEZ

40.

41.

42.

43.
44.

45.

beled LDL in homogenates: relation belween liver LDL receptors

and serum cholesterol in the fetus and post-morten. Biochim.
Biophys. Acta 836: 96-104.
MARKWELL,
M. A. K., HAAS,S. M., BIEBER,L. L. & TOLBERT,N. E.
(1986) A modification of ihe Lowry procedure lo simplify prolein
determination in membrane and lipoprolein samples. Anal. Bio
chem. 87: 206-210.
GOLDSTEIN,J. L., BASU,S. K. & BROWN,M. S. (1983) Recepior
mediated endocytosis of low density lipoprotein in cultured cells.
Meth. Enzymol. 98: 241-260.
FERNANDEZ,M. L. & MCNAMARA,D. }. (1989) High densily
lipoprolein (HDL) binding lo guinea pig hepalic membranes:
comparison of guinea pig and human ligands. Biochim. Biophys.
Acta 1042: 142-145.
SCATCHARD,
G. (1949) The attractions of proteins for small mol
ecules and ions. Ann. N.Y Acad. Sci. 50: 660-672.
STEEL,R. G. D. &TORRIE,J. H. (1960) Analysis of variance I. The
one way classification. In: Principles of Procedures for Statistics,
pp. 99-128, McGraw-Hill, New York, NY.
WILSON,J. N., WILSON,S. P. & EATON,R. P. (1984) Dietary fiber
and lipoprotein metabolism in the genetically obese Zucker rat.

Arteriosclerosis 4: 147-153.
46. BEYNEN,A. C. & KATAN,M. B. (1984) Relation belween ihe
response of serum cholesierol lo dielary choleslerol and to the
lype of dielary fat, prolein and fiber in hypo- and hyperresponsive
rais. Nutr. Rep. Int. 29: 1369-1381.
47. BEYNEN,A. C., VERSLUIS,
A., KATAN,M. B. & VANZUTPHEN,L. F.
M. (1987) Inleraclion of dietary cholesterol with the type of fat
and fiber with regard to plasma cholesterol response in rabbits.
Nutr. Rep. Int. 35: 355-360.
48. KRTTCHEVSKY,
D., TEPPER,S. A, SATCHITHANANDAM,
S., CASSIDY,
M. M. & VAHOUNY,G. V. (1988) Dietary fiber supplements:
effects on serum and liver lipids and on liver phospholipid com
position in rats. Lipids 23: 318-321.
49. MATHE,D., LUTTON,C., RAUTUREAU,
J., COSTE,T., GOUFFIER,E.,
SuLPiCE,J. C. &.CHEVALIER,
F. (1977) Effects of dietary fiber and
salt mixtures on ihe cholesterol metabolism of rats. /. Nutr. 107:
466-474.
50. LAFONT,H., LAIRON,D., VIGNE,J. L., CHANUSSOT,
F., CHABERT,C.,
PORTUGAL,
H., PAULI,A. M., CROTTE,C. & HAUTON,J. C. (1985)
Effect of wheal bran, pectin and cellulose on the secretion of bile
lipids in rats. /. Nutr. 115: 849-855.
51. O'BRIEN,R. C. & REISER,R. (1979) Comparative effects of puri
fied and human type diets on choleterol metabolism in ihe ral. /.
Nutr. 109: 98-104.
52. MIETTINEN,T. A. & TARPILA,S. (1977) Effecl of peclin on serum
cholesterol, fecal bile acids and biliary lipids in normolipidemic
and hyperlipidemic individuals. Clin. Chim. Acta 79: 471-477.
53. KAY,R. M. & TRUSWELL,
A. S. (1977) Effect of citrus pectin on
blood lipids and fecal steroid secretion in man. Am. f. Clin. Nutr.
30: 171-175.
54. YOUNT,N. Y. & MCNAMARA,D. J. (1988) Effect of dietary cho
lesterol vs cholestyramine on choleslerol homeosiasis in the
pregnant guinea pig and fetus. FASEB f. 2: A1215 (abs.).
55. OSTWALD,R. & FITCH,M. (1983) Guinea pig lipoproteins and
their changes in response to dietary cholesterol. In Methods in
Electrophoresis, pp. 133-137, CRC Press, Boca Raton, FL.
56. WITZUM,J. L., YOUNG,S. G., ELAM,R. L., CAREW,T. E. & FISHER,
M. (1985) Cholestyramine-induced changes in low density lipo
prolein composition and metabolism I. Studies in the guinea pig.
/. Lipid Res. 26: 92-103.
57. ILLMAN,R. J. & TOPPING,D. L. (1985) Effects of dietary oai bran
on faecal steroid excretion, plasma volatile fally acids and lipid
synlhesis in rats. Nutr. Res. 5: 839-846.
58. KRITCHEVSKY,
D., RYDER,E., FISHMAN,A., KAPLAN,M. & DEHOFF,
I. L. (1982) Influence of dietary fiber on food intake, feed effi
ciency and lipids in rats. Nutr. Rep. Int. 25: 783-787.
59. BOCHENECK,
W. J. & RODGERS,J. B. (1980) Comparison of hydrophobic surfactant and cholestyramine on lipid and sterol balance
in the rat. Exp. Mol. Pathol. 32: 223-230.
60. SHEPERD,
}. (1989) Mechanism of action of bile acid sequestranis
and olher lipid-lowering drugs. Cardiology 76: 65-74.
61. VAHOUNY,G. V, ROY,T, GALLO,L. L., STORY,J. A., KRITCHEVSKY,
D., CASSIDY,M., GRUND,B. M. & TREADWELL,
C. R. (1978) Dieiary fiber and lymphatic absorption of choleslerol in ihe rat. Am.
]. Clin. Nutr. 31: S203-S212.
62. KELLEY,
J. J. & TSAI,A. C. (1978) Effect of pectin, gum arabic and
agar on choleslerol absorption, synlhesis, and lurnover in rais. /.
Nur.108: 630-639.
63. SLATER,H. R., PACKARD,C. J., BICKER,S. & SHEPERD,J. (1980)
Effecls of cholestyramine on receptor-mediaied plasma clearance
and lissue upiake of human low density lipoproteins in ihe rabbit.
/. Biol. Chem. 255: 10210-10213.
64. SHEPERD,J., PACKARD,C. J., BICKER,S., LAWRIE,V. & MORGAN,H.
G. (1980) Cholestyramine promoles recepior-mediated
lowdensity-lipoproiein caiabolism. N. Engl. /. Med. 302: 1219-1222.

Downloaded from jn.nutrition.org by guest on October 9, 2011

26. IBRAHIM,
J. B. T. & McNAMARA,D. I. (1988) Cholesterol homeostasis in guinea pics fed saturated and polyunsaturated fat diets.
Biochim. Biophys. Acta 963: 109-118.
27. FERNANDEZ,
M. L. & MCNAMARA,D. J. (1989) Dietary fat medi
ated changes in hepatic apo B/E receptors in the guinea pig: effects
of polyunsaturated, monounsaturated and saturated fat. Metabo
lism 38: 1094-1102.
28. FERNANDEZ,
M. L. & MCNAMARA,D. J. (1989) Characterization
of HDL binding to guinea pig hepatic membranes: effects of
dietary fat quality and cholesterol feeding. FASEB f. 3:4231 (abs.|.
29. ALLAIN,C. G., POON,L. C. & CHON, C. G. S. (1974) Enzymatic
determination of total serum cholesterol. Clin. Chem. 20: 470475.
30. WARRICK,G. R., BERDERSOND,
J. & ALBERS,J. J. (1982) Dexiransulphaie-Mg*2 precipitation procedure for quantitation of high
density lipoprotein cholesterol. Clin. Chem. 28: 1379-1388.
31. SALE,F. O., MARCHESIN!,
S., FISHMAN,P. H. & BERRA,B. (1984) A
sensitive enzymatic assay for determination of cholesterol in lipid
extracts. Anal. Biochem. 142: 347^50.
32. POUMAY,Y. & RoNVEAUX-DuvAL,
M. F. (1985) Rapid preparative
isolation of concentrated low density lipoproteins and of lipoprotein-deficient serum using vertical rotor gradient ultracentrifugation. /. Lipid Res. 26: 1476-1480.
33. LINDGREN,F. T. (1975) Preparative ultracentrifugai laboratory
procedures for suggestions for lipoprotein analysis. In: Analysis
of Lipids and Lipoproteins (Perkins, E. G., ed), pp. 204-224,
American Oil Chemists Society, Champaign, II.
34. LAEMMLI,
U. K. (1974) Cleaveage of structural proteins during
the assembly of the head of the bacteriophage T4. Nature 227:
680-685.
35. NORDSTROM,J. L., RODWELL,V. W. & MTTSCHELEN,
J. J. (1977)
Interconversion of active and inactive forms of rat liver hydroxymethyl-glutaryl CoA reduc-ase/. Biol. Chem. 252: 8924-8934.
36. MCNAMARA,D. J., QUACKENBUSH,
F. W. & RODWELL,
V. W. (1972)
Regulation of hepatic 3-hydroxy-3-methylglutaryl
Coenzyme A
reduc-ase./. Biol. Chem. 247: 5805-5810.
37. SHAPIRO,D. f., NORDSTROM,
J. L., MITSCHELEN,
J. J., RODWELL,
V. W.
& SCHIMKE,R. T. (1974) Micro assay for 3-hydroxy-3-methyl
glutaryl CoA reduc-ase in ral liver and in L-cell fibroblasi. Bio
chim. Biophys. Acta 370: 369-377.
38. KOVANEN,P. T., BROWN,M. S. & GOLDSTEIN,J. L. (1979) in
creased binding of low density lipoprotein to liver membranes
from rats treated with a 17-a ethynil estradici. /. Biol. Chem. 254:
11367-11373.
39. RUDLING,M. J. & PETERSON,C. O. (1985) LDL receptors in
bovine tissues assayed as the heparin-sensitive binding of 125I-la-

ET AL.