Lipids

Pectin Isolated From Prickly Pear (Opuntia sp.)Modifies Low Density Lipoprotein Metabolism inCholesterol-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

ABSTRACT The effect of prickly pear soluble fiber onlow density lipoprotein (LDL) metabolism was investigated by feeding male guinea pigs either a nonpurifieddiet containing 0.25% cholesterol (HC diet) or the HCdiet + 1% prickly pear pectin (HC-P diet). Plasmacholesterol levels were significantly decreased by theHC-P diet, with a 33% decrease in LDL levels (p E

oCE

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io-B

e'42nf**,o

Ai A-\*i\\i'\O

' oi//

\,*-

U^'"-o-oi5*0

0HC.....HC_pli?

"* O~- 0 O-o 0-00O.- ---.. ... -.. -.. 9m

1.000 1.025 1.050 1.075 1.100 1.125 1.150 1.175

DENSITY (g/ml)

FIGURE 2 Typical density fractionation profiles of plasmalipoproteins of guinea pigs fed either the HC (O) or HC-P ()diet. Plasma lipoproteins were fractionated by density gradient ultracentrifugation in a VC-53 rotor as described byPoumay and Ronveaux-Duval (32).

tion of 10 u/mL, I25I-LDLbinding values were 459.8 91.0 (n - 10) vs. 792.7 225.2 (n = 11) ng bound/mgmembrane protein for guinea pigs fed the HC and theHC-P diets, respectively (p < 0.001). A significant negative correlation (r = -0.597, p < 0.005 was found betweenplasma LDL cholesterol levels and receptor-mediatedLDL binding to guinea pig hepatic membranes for animals fed the HC and HC-P diets (Fig. 3).

A typical saturation curve for the binding of LDL toguinea pig hepatic membranes for animals fed the HC-Pdiet is shown in Figure 4. Total binding increased withincreasing concentrations of 125I-LDL,nonspecific binding exhibited linearity and specific binding reached saturation at a concentration of 20 ug/mL.

Concentration-dependent binding curves for thebinding of LDL to guinea pig hepatic membranes fromboth groups of animals are shown in Figure 5. Bothmembrane preparations exhibited saturation at 20ug/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

DietHC

HC-PTotal6.11.9"

2.6 0.8"Cholesterol2Freemg/g4.11.3*

2.3 0.6"Ester2.00.9a

0.3 0.3bHMG-CoA

Reduc-ase3pmol/(min-mg

protein)2.03

0.321.99 0.31

'Data are presented as means so. Values in the same column with different superscripts are significantly different (p < 0.002) as assessed byANOVA 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.

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PECTIN AND LDL METABOLISM 1287

O)E

\enc

X

O)C

TDCm_iQ

1200-

900

600

300

O HC DlatHC-P Diet

O 10 20 30 40 SO 60 70 80 90 100 110

LDL Cholesterol (mg/100 mL)FIGURE 3 Regression analysis of relationship between

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

5. A 1.5-fold increase in the apo B/E receptor Bmaxvaluewas observed for hepatic membranes from guinea pigsfed the HC-P diet as compared to animals fed the HCdiet (2137 vs. 1300 ng/mg, respectively) whereas theaffinity constants for the apo B/E receptor for LDLwerethe same for both membrane preparations (18.5vs. 16.1ug/mL) (Fig. 5). The equilibrium parameters of KEoC

O

m 250-m_io

O 10 20 30LDL CONCENTRATION

FIGURE 5 Binding kinetics and Scatchard plots (inset) forreceptor-mediated binding (at 37C)of guinea pig LDL tohepatic 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, andBmaxvalues were 1300 and 2137 ng/mg protein for the animalsfed the HC and HC-P diets, respectively.

sources, including citrus pectin (45, 46) and other commercial brands (16, 47), has been consistently found toreduce plasma cholesterol levels in animal modelswhen fed at levels ranging from 5 to 20% (13, 15,45,46,48, 49). This cholesterol-lowering effect has been observed even in the presence of high fat (50) and highcholesterol diets (47, 51). In clinical studies, pectin hasalso shown a hypocholesterolemic effect in normal andhyperlipidemic subjects (52, 53).

Similarly, we found that prickly pear pectin had ahypocholesterolemic effect that affected both LDL andHDL fractions when fed at a concentration as low as 1%.Ney et al. (16) reported a normalization of rat plasmalipoproteins from inclusion of oat bran in a hypercho-lesterolemic atherogenic diet; the major effect was inthe VLDL fraction. A decrease in the LDL and HDLcholesterol was also reported, though apo A-I production was not affected. An increase in both LDLand HDLplasma cholesterol levels has been documented inguinea pigs fed a 0.25% cholesterol diet when comparedto animals fed nonpurified diets (28). In the presentstudy, adding pectin to the HC diet had a hypocholesterolemic effect in guinea pigs that resulted innormalization of plasma LDL and HDL cholesterollevels.

The hypocholesterolemic effect of pectin was notconsistent in all guinea pigs: some of the animals fed theHC-P diet had normal plasma cholesterol values of

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1288 FERNANDEZ ET AL.

TABLE 3

Equilibrium constants for LDL binding to guinea pig hepaticmembranes from animals fed nonpurified diet + 0.25% cholesterol

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

Diet2 Bmax

\ig/mL ng/mg protein

HCHC-P

19.817.5

7.34.2

16282385

316'342b

'Data are presented as means so. Values with different superscripts in the same column are signfiicantly different (p < 0.02) asassessed by ANOVA and the least significant difference test.

2n = 4 hepatic membrane preparations per group.

30-40 mg/100 mL reported for guinea pigs fed a non-purified diet whereas others had values of 50-70 mg/100mL, which are typical of guinea pigs fed the HC diet. Ithas been found in this and other studies that the plasmacholesterol levels for guinea pigs fed a high cholesteroldiet range from 50 to 140 mg/100 mL (28,54), suggestingthat some guinea pigs are hyper-responders to the HCdiet and others are not. A similar effect was observedwith the addition of pectin to the HC diet, in that someguinea pigs had a more pronounced response than othersto prickly pear pectin in the presence of the high cholesterol diet. On average, an overall 26% reduction in totalcholesterol and 33% reduction in LDL and HDL cholesterol were observed (n = 11). Regression analysis of therelationship between plasma LDL cholesterol levels andLDL receptor binding indicated that there was a significant negative correlation (p < 0.005) between these twoparameters. The observed correlation is consistent withthe hypothesis that apo B/E receptor expression is amajor determinant of plasma LDL cholesterol levels(41). Because pectin was added to the HC diet at aconcentration of only 1%, it would be of interest todetermine the hypocholesterolemic response to pricklypear pectin when fed at higher concentrations of 5-15%,as used for studies of other fibers by various investigators.

Prickly pear pectin effects on LDL density. As previously reported for guinea pigs fed different dietary fats(27), plasma from both groups of animals exhibited LDLheterogeneity characterized by the presence of LDLsubfractions (Fig. 2). It has been observed that LDLdensity decreases to lower values when guinea pigs arefed high cholesterol diets when compared to animals fednonpurified diets with low concentrations of dietarycholesterol (55). Adding pectin to the HC diet shiftedthe LDL peak density from 1.040 to 1.055 g/mL (Fig. 2),approaching values reported for guinea pigs fed non-purified diets and heading toward the values of 1.07-1.08 g/mL reported for animals fed 7.5% dietary fat (26,27).

ryl

aid

These data suggest that consumption of pectin decreases the size and increases the density of LDL. Anincrease in LDL peak density also results from consumption of polyunsaturated versus saturated fat diets(26, 27), and a decrease in LDL particle size results fromthe intake of cholestyramine (56). Because larger, lessdense LDL particles contain more cholesteryl ester (27,56), it would be predicted that pectin reduces cholesterylester concentrations of plasma LDL.

Pectin effects on hepatic cholesterol synthesis ancholesterol content. Adding 0.25% cholesterol to thediet reduced guinea pig hepatic cholesterogenesis by90%. Values for hepatic HMG-CoA reduc-ase activityfor animals fed a nonpurified diet are 30-40 pmol/(minmg protein) (N. Y. Yount and D. J. McNamara, unpublished data). Adding pectin to the cholesterol diet didnot affect hepatic cholesterol synthesis since bothgroups of animals had similar levels of HMG-CoA re-ductase activity. Reports concerning the effects of oatbran and pectin on hepatic HMG-CoA reduc-ase arecontradictory. Inclusion of oat bran decreased HMG-CoA reduc-ase levels when compared to levels with asemipurified control diet (22), and no effect on hepaticHMG-CoA reduc-asewas observed by addition of cellulose 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 semi-purified diet. Illman et al. (57) compared the effects ofoat bran and cellulose on hepalic cholesterol synthesisin rats; synlhesis was measured by incorporalion of[3H]water inlo digilonin-precipilable sierols. Theyfound a significanl reduction in hepatic cholesterol synthesis in animals fed the cellulose diet. In this study,adding pectin did not modify the levels of hepatic HMG-CoA reduc-ase activity that were almost completelysuppressed by the 0.25 % cholesterol diet.

Many investigalors have reponed a decrease in livercholesterol content from the addilion of peclin lo diels(45, 48, 58). In ihis report a similar response was observed in lhal prickly pear peclin significanlly reducedhepalic total, free and esterified choleslerol levels whencompared lo levels wilh ihe HC diel (p < 0.001) (Table2). Like peclin, choleslyramine added lo a high choleslerol diel also reduced hepalic choleslerol in rals (59).

Effect of pectin on LDL binding to hepatic membranes. The significanl increase in LDL binding observed for hepalic membranes from animals fed iheHC-P diel suggesls lhal iwo possible mechanisms maybe involved. One is an increase of receplor affinily forihe LDL particle, and ihe second is an increase in apoB/E receplor number. Scalchard analysis demonslraledlhal ihe Kd values were ihe same for bolh membranepreparalions whereas Bmaxwas 1.5-fold higher for guineapigs fed ihe HC-P diel, indicaling lhal ihe number of apoB/E recepiors was significanlly increased.

Comparative effects of cholestyramine and pricklypear pectin on cholesterol metabolism. Peclin and the

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PECTIN AND LDL METABOLISM 1289

bile acid squestrant resin cholestyramine appear tohave similar mechanisms in reducing plasma cholesterol levels. Cholestyramine binds bile acids in theintestine and interrupts their enterohepatic circulation.As a result of this interruption, three enzymes areinduced: phosphatidic acid phosphatase, cholesterol 7cc-hydroxylase and HMG-CoA reductase (60). Consequently, an increase in triglycrides, an increaseddemand for intracellular cholesterol and an increasedcholesterol synthesis would be expected.

Similar to what has been reported for cholestyramine(60, 61), prickly pear pectin may also affect cholesterolabsorption, as has been documented for citrus pectin(62), and may increase bile acid fecal excretion, as hasbeen reported for some commercial pectin sources inclinical (52) and animal studies (13).

Cholestyramine has been shown to reduce plasmatotal and LDL cholesterol levels (63, 64), decrease cholesterol hepatic content (59), decrease LDL particle sizeand increase LDL peak density (56); increase receptor-mediated plasma clearance in rabbits (63) and humans(64); and increase LDL binding to guinea pig hepaticmembranes (56). These observations are similar to whathas been reported in this study using prickly pear pectin.Contrary to what has been reported for rats fed a highcholesterol diet treated with cholestyramine (59),prickly pear pectin did not increase hepatic HMG-CoAreductase levels.

In summary, adding 1% prickly pear pectin to a highcholesterol diet decreased total, LDL and HDL cholesterol levels (p < 0.02); altered LDL particle size as demonstrated by an increase in LDL peak density; decreasedhepatic concentrations of total, free and esterified cholesterol (p < 0.002); did not change hepatic HMG-CoAreductase activity; and significantly increased the number of hepatic apo B/E receptors (p < 0.001) withoutmodifying the receptor affinity. From these observations, we conclude that the mechanisms involved in theplasma hypocholesterolemic effect of prickly pear pectin could be analogous to those reported for cholestyramine and other bile acid-binding resins in clinicaland animal studies (59, 63, 64).

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