The Journal of Nutrition Vol. 128 No. 9 September 1998, pp. 1434-1441

Dietary Soluble Fiber Lowers Plasma LDL Cholesterol Concentrations by Altering Lipoprotein Metabolism in Female Guinea Pigs^1,2

Hong Shen, Lin He, Ralph L. Price, and Maria Luz Fernandez³

Lipid Metabolism Laboratory, Department of Nutritional Sciences, University of Arizona, Tucson, AZ 85721

	ABSTRACT

Abstract Introduction Methods Results Discussion References

This experiment was designed to evaluate the effects of pectin (PE), guar gum (GG) and psyllium (PSY) intake on VLDL and LDL metabolism in female guinea pigs fed high dietary cholesterol. Guinea pigs were fed a 15 g/100 g fat diet containing 0.25 g/100 g cholesterol with 12.5 g/100 g PE, 12.5 g/100 g GG, 7.5 g/100 g PSY or 12.5 g/100 g cellulose (control diet) for 4 wk. Plasma cholesterol concentrations were 29, 43 and 39% lower in guinea pigs fed PE, GG or PSY, respectively, compared with the control group (P < 0.0001). Plasma apolipoprotein (apo) B concentrations were 16-22% lower in the groups fed soluble fiber compared with the control group (P < 0.01). In contrast, hepatic cholesterol and triglyceride concentrations were not different among the PE, GG, PSY and control groups. No differences in triacylglycerol (TAG) or apo B secretion rates, measured by blocking VLDL catabolism by triton (WR 1339) injection, were observed, whereas plasma LDL apo B fractional catabolic rates (FCR), determined by injection of radiolabeled LDL, were higher in guinea pigs fed GG or PSY than in those from the control group. All sources of dietary soluble fiber reduced LDL apo B flux (P < 0.05). These results suggest that the mechanisms of plasma LDL cholesterol lowering by dietary soluble fiber are distinctive for each fiber source and result in specific alterations in lipoprotein metabolism in female guinea pigs. Differences between male and female guinea pigs in response to these diets are discussed.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Over the last few years, studies have focused mainly on the effect of soluble fiber on male subjects, whereas little is known about the effect of soluble fiber on female subjects. Numerous studies have shown that men and women present different characteristics in developing cardiovascular disease and increasing plasma lipid levels (Monique-Verschuren & Kromhout 1995, Nikkila et al. 1996, Wenger 1995). Men are more likely to develop coronary heart disease than premenopausal women (Heller and Jacobs 1978). In addition, women are more likely to be influenced by a "Western" diet, known for its high fat, high cholesterol and excess energy (Bush et al. 1988). Thus it is essential to study the effects of soluble dietary fiber on female lipid metabolism, particularly under high cholesterol challenge.

Recently, studies in which the effects of dietary fiber on differences in plasma-cholesterol lowering between men and women were evaluated, men were more responsive than postmenopausal women in lowering plasma LDL cholesterol and apolipoprotein B (apo B)⁴ concentrations (Jenkins et al. 1993). In addition, reported data on guinea pigs demonstrate that females are more susceptible than males to a hypercholesterolemic diet; thus the cholesterol-lowering properties of dietary soluble fiber are not as effective as with male guinea pigs (Fernandez et al. 1995c).

This study was performed to determine some of the mechanisms responsible for the plasma cholesterol lowering previously observed in female guinea pigs fed pectin, guar gum and psyllium (Fernandez et al. 1995c) and to evaluate whether those mechanisms are different from our reports in male guinea pigs (Fernandez 1995 and Fernandez et al. 1997). Guinea pigs were chosen in this and previous investigations (Fernandez 1995, Fernandez et al. 1994, 1995a, 1995b and 1995c), because, like humans, they transport cholesterol mainly in LDL (Fernandez 1995), they have similar distribution of hepatic cholesterol pools with higher concentrations of free vs. esterified cholesterol (Angelin et al. 1992) and they have plasma cholesteryl ester transfer protein (CETP) activity, which makes the intravascular processing of lipoproteins analogous to that of humans (Ha and Barter 1982). In addition, dietary fiber lowers plasma LDL cholesterol concentrations in guinea pigs as in humans; this has been documented in numerous reports from our laboratory (Fernandez 1995, Fernandez et al. 1990, 1994, 1995a, 1995b, 1995c and 1997, Vidal-Quintanar et al. 1997). All of these characteristics make the guinea pig an appropriate animal model with which to evaluate the effects of dietary soluble fiber on the mechanisms of plasma LDL cholesterol lowering.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Materials.  Reagents were obtained from the following sources: Tyloxapol (Triton WR-1339) and triacylglycerol enzymatic kits, rapid silver staining electrophoresis kit and cyanogen bromide activated sepharose were purchased from Sigma Chemical (St. Louis, MO). The cholesterol enzymatic assay kit, cholesterol oxidase and cholesterol esterase were from Boehringer-Mannheim (Indianapolis, IN), free cholesterol and phospholipid enzymatic assay kits from Waco Pure Chemical Industries (Osaka, Japan) and halothane from Halocarbon (Hackensack, NJ). ¹²⁵I was from Amersham (Arlington Heights, IL), ¹³¹I from NEN (Boston, MA) and quickseal ultracentrifugation tubes from Beckman Instruments (Palo Alto, CA). The radial immunodiffusion kit was from Bio-Rad (Richmond, CA). High methoxylated pectin made from lime peels and containing 6.7% methoxyl groups and 74% galacturonic acid was obtained from Grinsted Products (Industrial Airport, KS); guar gum type MMM/12 containing 84-89% fiber, 10% protein and 1.5% ash was provided by Meer Corporation (North Bergen, NJ); powdered psyllium husks #40 purified 95% containing <3% fat and 1% protein were from Meer Corporation.

Diets.  Diets were prepared by Research Diets (New Brunswick, NJ). The four diets had the same composition except for the fiber source, as indicated in Table 1. The fiber source was 12.5 g/100 g cellulose (control diet, CNT), 12.5 g/100 g pectin (PE), 12.5 g/100 g guar gum (GG) or 7.5 g/100 g psyllium plus 5 g/100 g cellulose (PSY) (Table 1). Psyllium was used at a lower concentration than the other sources of soluble fiber because previous reports have demonstrated that this concentration results in similar hypocholesterolemic responses (Fernandez 1995, Fernandez et al. 1997). Diets contained 15 g/100 g palm oil (16:0, 43.3%; 18:0, 4.1%; 18:1, 39.8%; 18:2, 9.7%) and fat represented 35% of the energy content. The amount of cholesterol was 0.25 g/100 g. This dietary cholesterol concentration was chosen to define the effects of soluble fiber intake on VLDL metabolism when the amount of absorbed dietary cholesterol is equivalent to 1.5 times the daily endogenous cholesterol synthesis rate of guinea pigs (Lin et al. 1992).

Animals.  Female Hartley guinea pigs (Sasco Sprague Dawley, Omaha, NE) weighing 300-400 g were randomly assigned to one of four dietary groups (n = 6) for 4 wk to achieve a metabolic steady state before analysis. They were housed in a room with controlled lighting (light 0700 to 1900 h) and consumed feed and water ad libitum. Guinea pigs used for the isolation and characterization of VLDL and LDL (in vitro studies) and for measurement of triacylglycerol (TAG) secretion rate were killed by heart puncture after halothane anesthesia. Guinea pigs used for in vivo LDL kinetic studies were killed by an excess of halothane vapors. All animal experiments were conducted in accordance with U.S. Public Health Service/U.S. Department of Agriculture guidelines, and experimental procedures were approved by the University of Arizona Institutional Animal Care and Use Committee.

Plasma and microsome preparation.  Plasma was separated by centrifugation at 2000 × g for analysis of cholesterol, TAG and apo B and stored at 4°C. A mixture of aprotonin, phenylmethylsulfonilfluoride and sodium azide was added to plasma samples to minimize changes in lipoprotein composition during isolation.

Liver tissue was passed through a tissue grinder into 1:2.5 (wt/v) homogenization buffer (50 mmol/L KH₂PO₄, 0.1 mol/L sucrose, 50 mmol/L KCl, 30 mmol/L EDTA, 50 mmol/L NaCl and 2 mmol/L dithiothreitol, pH 7.2) and homogenized using a Potter-Elvehjem homogenizer. Microsome pellets were obtained after two 15-min centrifugations at 10,000 × g and two ultracentrifugations at 100,000 × g at 4°C. Pellets were resuspended in buffer and kept at 70°C.

Plasma and hepatic lipids.  Plasma total cholesterol, HDL cholesterol and TAG concentrations were determined by enzymatic methods (Allain et al. 1974). HDL cholesterol was measured after precipitation of apo B-containing lipoproteins with MgCl₂-dextran sulfate (Warnick et al. 1982).

Hepatic lipids were separated by resuspending liver in chloroform/methanol (2:1). After resuspension in methanol, total choleterol, free cholesterol and TAG were determined (Carr et al. 1993). Esterified cholesterol was calculated by subtracting free from total hepatic cholesterol. Microsomal lipids were also isolated by chloroform/methanol (2:1); after resuspension in 1 g/100 g triton solution, free cholesterol and phospholipid were measured by enzymatic methods (Carr et al. 1993).

Triacylglycerol secretion rate.  The rates of VLDL TAG and apo B secretion were determined by blocking VLDL catabolism with Triton WR-1339, a non-ionic detergent that coats VLDL particles, blocking the action of lipoprotein lipase (LPL) in vivo and thus preventing VLDL catabolism. Food was removed from guinea pigs 12 h before surgery, and a catheter was inserted into the carotid artery for injection of Triton and continuous plasma sampling. Animals were deprived of food during the 8 h of the experiment to ensure that the measured plasma TAG levels reflected VLDL secretion and not influx of dietary TAG as chylomicrons. A 20 g/100 g triton solution (100 mg/kg of body weight) was injected and blood samples (0.4 mL) were taken at 0, 5, 10, 15, 20, 35, 50, 75, 120, 180, 300 and 480 min. Plasma was separated from red blood cells and TAG concentrations were measured for each time point. TAG accumulation in plasma increased linearly with time (r = 0.99). TAG secretion rate was calculated by regression analysis as mg TAG secreted/(kg body weight·h). Apo B secretion rate was calculated as [VLDL TAG secretion rate × apo B concentration (%)]/VLDL TAG (%).

Isolation and characterization of nascent VLDL, mature VLDL and LDL.  Plasma and VLDL cholesterol were determined by enzymatic methods (Allain et al. 1974). At the end of the TAG secretion rate experiments, animals were killed and nascent VLDL was isolated (d = 1.006 kg/L); mature VLDL was isolated at d < 1.019 kg/L, a density fraction including intermediate density lipoprotein, which is present in negligible amounts in guinea pigs (Fernandez et al. 1993), and LDL at d = 1.019-1.09 kg/L. Lipoprotein composition was determined by measuring protein (Markwell et al. 1978), TAG, phospholipids and cholesterol. VLDL apo B was selectively precipitated with isopropyl alcohol (Egusa et al. 1987); total VLDL protein was determined in the supernatant and VLDL apo B calculated by difference. The number of component molecules of nascent VLDL, mature VLDL and LDL was calculated assuming one apo B per lipoprotein particle with a molecular weight of 412,000 as reported for guinea pigs (Chapman et al. 1975). The molecular weight of TAG, free and esterified cholesterol and phospholipids was calculated as 885.4, 386.6, 646 and 734 g/mol, respectively.

Determination of apo B concentrations in plasma.  Polyclonal antibodies against apo B-100 were prepared by injecting purified guinea pig LDL (checked by SDS PAGE) into a sheep in one dose (300 mg/L) followed by two booster doses (0.2 mg/L) every 10 d. Apo B antibodies were purified by an antigen affinity column and were eluted by modification of pH (Ishida and Paigen 1992). Concentrations of apo B in plasma were measured by silver enhanced radioimmunodiffusion. Standards were prepared from guinea pig LDL isolated by ultracentrifugation at a narrow density (d = 1.023-1.075 kg/L) and further purified by agarose affinity chromatography (Fernandez 1995). Linear regression correlations were used to calculate sample concentrations.

Plasma lipoprotein isolation and labeling.  Pooled LDL from guinea pigs fed control, PE, GG and PSY diets were separated by sequential ultracentrifugation in a L8-M ultracentrifuge (Beckman Instruments) at 125,000 × g at 15°C for 19 h in a Ti50 rotor at a density range of 1.02-1.09 kg/L for LDL to be injected into guinea pigs fed the homologous diet. LDL were dialyzed against 0.09% NaCl and 0.01% EDTA for 24 h. Purity of LDL preparations was checked by electrophoresis (data not shown).

In vivo VLDL and LDL kinetics.  LDL were iodinated according to Goldstein et al. (1983) with ¹²⁵I and used within 2 d of iodination to minimize possible changes due to radiation oxidation (Khuow et al. 1993). Calculations of LDL-associated radioactivity indicated that the percentage of radioactivity associated with apo B was 95%.

Guinea pigs were injected with radiolabeled LDL through an indwelling catheter via the carotid artery, and plasma samples were taken at 5 min (baseline), 0.5, 1, 3, 5, 10, 22 and 28 h. The disappearance in plasma of radiolabeled LDL was followed by counting plasma samples directly in the gamma counter; LDL fractional catabolic rates (FCR) were determined by use of a two-pool model as described by Matthews (1957). Apo B protein mass was measured as indicated above. LDL apo B pool size was calculated by multiplying apo B concentration in mg/dL× plasma volume adjusted to 1 kg. Plasma volume was assumed to be 4.0% of guinea pig weight as previously reported (Fernandez 1995). LDL apo B flux was also calculated by multiplying apo B pool size [mg/(kg·h)] × FCR (h¹).

Statistical analysis.  One-way ANOVA was used to determine differences in plasma cholesterol, TAG concentrations, TAG secretion rate, nascent and mature VLDL composition, LDL apo B turnover and apo B flux among the control and the three dietary soluble fiber groups in female guinea pigs. The Newman-Keuls method was used as a post-hoc test (INSTAT, San Diego, CA). Statistical analysis of the kinetic model data were best fitted using a two-pool model (JANA, SCI Software, Lexington, KY).

	RESULTS

Abstract Introduction Methods Results Discussion References

PE, GG and PSY effects on plasma lipids in female guinea pigs.  There were no significant differences in body weights among female guinea pigs fed the different diets, which indicates that they consumed similar amounts of nutrients (data not shown). Plasma cholesterol concentrations were lower than that in the control group in groups fed PE, GG and PSY by 29, 43 and 39%, respectively (P < 0.0001) (Table 2). Plasma TAG concentrations were reduced (22%) only in the PE group (P < 0.01), whereas plasma apo B concentrations were 16-22% lower in the soluble fiber groups compared with the control group (P < 0.01) (Table 2). In addition PE, GG and PSY intake resulted in lower plasma LDL cholesterol concentrations by 40-55%, whereas plasma VLDL and HDL cholesterol were not affected by the dietary treatments (Table 3).

Mature VLDL composition was affected only by PSY (Table 4). Guinea pigs fed the PSY diet had a higher relative proportion of TAG in VLDL compared with VLDL isolated from control, PE- and GG-fed guinea pigs (P < 0.001). No other compositional differences were observed in VLDL from the four dietary groups. Similarly, the number of TAG molecules was higher in LDL derived from guinea pigs fed the PSY diet (Fig. 1). In addition, the number of PL molecules was greater in this group. LDL from control, PE and GG groups had compositional and size characteristics that did not differ.

View larger version (31K): [in this window] [in a new window]   Fig 1. Numbers of phospholipid (PL), triacylglycerol (TAG), free cholesterol (FC), and cholesteryl ester (CE) molecules of LDL particles from female guinea pigs fed control (CNT), pectin (PE), guar gum (GG) or psyllium (PSY) diets. Values are means ± SD (of total molecules), n = 6. *Indicates significantly different from the control diet (P < 0.01).

PE, GG and SPY effects on hepatic lipids in female guinea pigs.  PE, GG or PSY intake did not result in differences in hepatic cholesterol or TAG concentrations compared with guinea pigs fed the control diet. Hepatic cholesterol concentrations were 9.1 ± 2.0, 11.9 ± 4.1, 8.3 ± 1.8 and 7.8 ± 2.0 µmol/g for control, PE-, GG- and PSY-fed guinea pigs, respectively. Hepatic triglyceride values were 24.5 ± 8.3, 26.8 ± 9.9, 16.8 ± 6.0 and 16.2 ± 5.0 µmol/g for control, PE-, GG- and PSY-fed guinea pigs, respectively. However, PE and PSY increased phospholipids in hepatic microsomes compared with controls (P < 0.01) (Table 5). Guinea pigs consuming the three fiber sources had a free cholesterol/phospholipid ratio lower than that of controls (Table 5), suggesting that soluble fiber may have changed the fluidity of the cell membranes and passage of substances into and out of the cells.

PE, GG and PSY effects on VLDL TAG and apo B secretion in female guinea pigs.  To determine possible sites of action of PE, GG and PSY, TAG and apo B secretion rates were measured in female guinea pigs. There were no significant differences in TAG or apo B secretion rates among groups (Table 6). However, the composition of nascent VLDL isolated from soluble fiber-fed guinea pigs was affected by PE, GG and PSY. Guinea pigs fed the soluble fiber diets secreted larger VLDL containing more TAG and phospholipid molecules than the controls (Fig. 2). These compositional and size modifications in nascent VLDL might be related to different metabolic fates in the plasma compartment.

View larger version (39K): [in this window] [in a new window]   Fig 2. Numbers of phospholipid (PL), triacylglycerol (TAG), free cholesterol (FC) and cholesteryl ester (CE) molecules of nascent VLDL particles from female guinea pigs fed control (CNT), pectin (PE), guar gum (GG) and psyllium (PSY) diets. Values are means ± SD (of total molecules), n = 6. *Indicates significantly different from the control diet (P < 0.01).

PE, GG and PSY effects on LDL kinetics in female guinea pigs.  LDL kinetic parameters were affected by soluble fiber as indicated in Table 7. Apo B pool size was lower in the soluble fiber groups, consistent with the reduced plasma LDL cholesterol and apo B concentrations. Guinea pigs fed GG and PSY had faster LDL Apo B FCR than controls (P < 0.01) (Table 7), whereas the PE and control groups did not differ. LDL apo B flux was lower in the PE, GG and PSY groups than in controls (P < 0.05).

Gender differences in response to dietary fiber and dietary cholesterol.  For the purpose of comparing gender differences in response to dietary soluble fiber, some of the data obtained from this study were compared with data published for male guinea pigs (Fernandez 1995, Fernandez et al. 1997). Male and female guinea pigs respond differently to high cholesterol intake as we had observed previously (Fernandez et al. 1995c). High cholesterol caused higher plasma cholesterol concentrations in female compared with male guinea pigs (Fig. 3), although PE, GG and PSY lowered plasma cholesterol to the same extent compared with control animals. In addition, females had lower hepatic cholesterol than males (Fig. 4). Although PE, GG and PSY intake resulted in lower hepatic cholesterol in males, this was not the case for female guinea pigs. PE had the greatest effect in male guinea pigs, whereas there was a tendency for PE intake to result in the highest hepatic cholesterol (P = 0.08) in females, suggesting a gender difference in the mechanisms of plasma cholesterol lowering by PE (Fig. 4). In addition, TAG secretion rates were higher for males (Fig. 5) and LDL apo B FCR rates were higher in females (Fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig 3. Comparisons of dietary fiber effects on plasma total cholesterol concentrations in male (adapted from Fernandez et al. 1997) and female guinea pigs from this study (Table 2). Bars represent the mean ± SD of males or females, n = 18. Diets are control (CNT), pectin (PE), guar gum (GG) or psyllium (PSY). *Indicates significantly different from control (P < 0.01).

View larger version (18K): [in this window] [in a new window]   Fig 4. Comparisons of dietary fiber effects on hepatic cholesterol concentrations in male (adapted from Fernandez et al. 1997) and female guinea pigs from this study (see text). Bars represent the mean ± SD of males or females, n = 6. Diets are control (CNT) pectin (PE), guar gum (GG) or psyllium (PSY). *Indicates significantly different from control.

View larger version (22K): [in this window] [in a new window]   Fig 5. Comparisons of dietary fiber effects on triacylglycerol secretion rates in male (adapted from Fernandez et al. 1997) and female guinea pigs from this study (Table 6). Bars represent the mean ± SD of males or females, n = 6. Diets are control (CNT), pectin (PE), guar gum (GG) or psyllium (PSY).

View larger version (18K): [in this window] [in a new window]   Fig 6. Comparisons of dietary fiber effects on LDL apo B fractional catabolic rates (FCR) in male (adapted from Fernandez et al. 1997) and female guinea pigs from this study. Bars represent the mean ± SD of males or females, n = 6. Diets are control (CNT), pectin (PE), guar gum (GG) or psyllium (PSY). *Indicates significantly different from control (P < 0.01).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Although PE, GG and PSY decrease plasma cholesterol levels in humans and some animal models, little is known about the effects of soluble fiber on females, especially how it affects female lipoprotein metabolism. There has been some interest in determining whether men and women respond differently to dietary interventions (Bush et al. 1988), but very few studies have addressed how dietary fiber may affect the potential mechanisms of plasma lipid lowering. It was therefore of interest to assess how different sources of soluble fiber influence lipoprotein metabolism in female guinea pigs.

The results from this study indicated that the plasma cholesterol-lowering effects of soluble fiber (PE, GG or PSY) with intake of high dietary cholesterol were achieved by reducing LDL cholesterol levels and that there were specific mechanisms of plasma cholesterol lowering associated with the type of dietary fiber.

PE, GG and PSY effects on female guinea pigs.  High dietary cholesterol resulted in elevated plasma cholesterol in female guinea pigs, which was consistent with previous studies (Fernandez et al. 1995c) suggesting that guinea pigs, like hamsters, are sensitive to high dietary cholesterol. The elevated plasma cholesterol concentrations appear to be related to suppression of hepatic LDL receptors (Lin et al. 1994, Spady and Dietschy 1988). In addition to their effects on plasma total and LDL cholesterol, PE, GG and PSY reduced plasma apo B concentrations, suggesting that soluble fiber reduces not only the amount of LDL cholesterol but also the number of LDL particles.

Hepatic lipids directly influence VLDL TAG and apo B secretion by the liver (Dixon and Ginsberg 1992). High cholesterol intake causes increased delivery of cholesterol to the liver through the chylomicron remnant and increased hepatic free and esterified cholesterol concentrations in guinea pigs (Fernandez 1995, Fernandez et al. 1995c). An increase in the intracellular mass of cholesteryl ester in cultured human Hep G2 cells results in stimulation of apo B secretion in the form of VLDL (Avramoglu et al. 1995, Dixon and Ginsberg 1992). In this study, soluble fiber did not reduce liver cholesterol in female guinea pigs, similar to our previous report (Fernandez et al. 1995c) and also did not affect apo B secretion rates. Although no effects on TAG or apo B secretion rates were observed as a result of diet, PE, GG and PSY did affect the composition of the secreted nascent VLDL, resulting in larger particles with higher numbers of TAG and PL molecules. These larger particles might affect the intravascular processing of VLDL and possibly be one of the factors contributing to the hypocholesterolemic action of soluble fiber.

Plasma LDL values are affected by modifications in VLDL metabolism, including the rate of conversion of VLDL to LDL. Basically, nascent VLDL are converted to mature VLDL and to LDL through the loss of TAG by the action of LPL. As TAG are lost, the concentration of cholesteryl ester (CE) increases, also through the action of CETP. In this study, the effects of PSY on mature VLDL composition were related to increases in TAG and decreases in CE, which are related to slowed conversion of VLDL to LDL (Nestel et al. 1983) and may contribute to the lower levels of plasma LDL cholesterol as a result of consuming PSY.

Plasma cholesterol concentrations are affected not only by VLDL compositional changes, but also by VLDL and LDL sizes. The densities of the particles decrease as the ratio of lipid to protein increases, and larger particles are associated with lower density. Based on the relationship among protein concentration, density and size, lipoprotein size can be estimated. There were no differences found in mature VLDL composition, which may be related to the lack of differences in the sizes of the particles in guinea pigs fed PE, GG or the control diet. Generally, large nascent VLDL will produce large mature VLDL. This was not the case in male guinea pigs where large nascent VLDL induced by dietary soluble fiber resulted in smaller mature VLDL than in the control animals (Fernandez et al. 1997) or in this study where the large nascent VLDL associated with dietary soluble fiber resulted in a VLDL particle that, except for the PSY group, had characteristics similar to that of the control group. These results might be associated with LPL and CETP activities. Data from male guinea pigs indicated that plasma CETP activity was lower in those fed soluble fiber (Fernandez et al. 1997). Although CETP activity was not measured in female guinea pigs, under most conditions, interventions that lower plasma levels of the apo B-containing lipoproteins will also lower plasma CETP activity (McNamara 1992).

Many studies have shown that dietary soluble fiber affects LDL apo B kinetics (Fernandez 1995, Mazur et al. 1990, Turley and Dietschy 1995). PSY intake has been demonstrated to decrease LDL production and increase LDL uptake in hamsters (Turley and Dietshcy 1995) and fermentable polysaccharides to increase LDL FCR in rats (Mazur et al. 1990). Higher numbers of hepatic apo B/E receptors and faster LDL FCR have been reported as a result of intake of PE, GG and PSY by male guinea pigs (Fernandez 1995, Fernandez et al. 1994, 1995a and 1995b). In this study, GG and PSY increased LDL FCR in female guinea pigs, which, in addition to the higher number of apo B/E receptors (Fernandez et al. 1995c), might relate to the smaller LDL particles because smaller LDL are more easily removed from plasma (Cryer et al. 1978). In addition, another important mechanism contributing to the plasma cholesterol lowering by dietary soluble fiber was the reduction in LDL apo B flux, which is related to a decreased conversion of VLDL to LDL. An unexpected finding in this study was that VLDL apo B secretion rates were lower than LDL apo B flux. The cause of this discrepancy is unknown at present and may be related to the use of different methods to calculate these parameters. Triton injection to block LPL action was used to determine apo B secretion rates, and radiolabeled isotopes were utilized to measure plasma LDL turnover. However, this discrepancy was not observed in our previous studies in male guinea pigs (Fernandez et al. 1997).

PE, GG and PSY intake resulted in moderate alterations in hepatic and microsomal lipids. Similar to our study, increases in hepatic TAG concentrations have been reported to result from intake of dietary cholesterol (Fungwe et al. 1993) and have been associated with decreased carnitine synthesis. This situation promotes fatty acid accumulation and TAG synthesis rather than shifting fatty acids for -oxidation (Fungwe et al. 1993). The increased hepatic TAG level may also be explained by reduced hepatic lipogenesis, reduced output or enhanced plasma clearance (Hexeberg et al. 1994). In this study, guinea pigs fed PE had the lowest values for TAG secretion and the highest hepatic TAG concentrations, although differences were not significant, which might suggest that the higher hepatic TAG could be due to a reduced secretion of TAG by the liver. In addition, PE and PSY increased PL levels in liver microsomes, whereas all three soluble fibers decreased the FC/PL ratio. Variations in this ratio are believed to influence the fluidity of the membrane and passage of substances into and out of the cell. A lower ratio of FC to PL increased hepatic microsome fluidity in rats and was associated with greater cholesterol excretion (Hotchgraf et al. 1997). Thus, it is possible that those changes in membrane fluidity mediated by soluble fiber observed in this study could be related to increased lipid transfer and greater rates of elimination from the body.

Gender differences in response to dietary fiber and high cholesterol.  Although the mechanisms of LDL cholesterol lowering of soluble fiber are different in male (Fernandez et al. 1997) and female guinea pigs, the hypocholesterolemic effects are comparable. However, female guinea pigs are more sensitive to dietary cholesterol and respond moderately to dietary soluble fiber in many aspects. In a human study, Jenkins et al. (1993) exchanged soluble fiber for insoluble fiber and kept all dietary components constant. There were significant reductions in plasma total, LDL and HDL cholesterol concentrations as well as apo B and apo A-I levels for all subjects. However, dietary soluble fiber had a greater proportional change in lowering plasma cholesterol and apo B levels in men than in women, similar to our observations in guinea pigs.

Studies have shown that males and females, especially when challenged with a high cholesterol diet, have different responses of plasma lipid metabolism. The major mechanisms associated with the different responses to dietary cholesterol are differences in the fractional absorption of dietary cholesterol and suppression of cholesterol synthesis in humans (McNamara et al. 1987) and, in the case of rats, an increase in bile acid synthesis and excretion (Moundras et al. 1994). Results from this study and our previously reported data on male guinea pigs (Fernandez 1995 and Fernandez et al. 1997) have shown that high dietary cholesterol induced higher plasma cholesterol and apo B levels in female than in male pigs, in agreement with the report of Van Vlijmen et al.(1996), who observed that female mice, when fed a high fat/cholesterol diet, had higher cholesterol levels than males.

The effects of dietary soluble fiber on hepatic cholesterol levels in males and females were different. In agreement with this study, soluble fiber did not lower hepatic cholesterol in female guinea pigs fed a high cholesterol diet (Fernandez et al. 1995c). The liver plays a central role in whole-body cholesterol homeostasis. Once cholesterol enters the hepatocyte, it may be catabolized to bile acids, excreted into bile as free cholesterol, secreted back into the plasma in lipoproteins or esterified by acyl CoA cholesterol acyltransferase (ACAT) and stored in the liver (Small 1988). The mechanisms regulating hepatic cholesterol homeostasis appear to be different in male and female guinea pigs (Fernandez et al. 1994, 1995a, 1995b, 1995c and 1997). In our previous studies, when male guinea pigs were fed high dietary cholesterol, PE, GG and PSY increased the activities of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase and cholesterol 7-hydroxylase and decreased ACAT activity (Fernandez et al. 1994, 1995a, 1995b). However, in female guinea pigs fed high dietary cholesterol, soluble fiber only marginally increased activities of HMG-CoA reductase and cholesterol 7-hydroxylase and marginally decreased activity of ACAT (Fernandez et al. 1995c). Dietary cholesterol seems to overwhelm the effects of the soluble fibers on hepatic enzymes in the case of females. These results indicate that the hypocholesterolemic effect produced by the soluble fibers may not be mediated via alterations in cholesterol degradation because of a decreased bile acid synthesis. It is possible that the effects of soluble fiber in females may be related more to increases in the excretion of fecal neutral sterols and decreases in cholesterol reabsorption. Recent reports have shown that viscous soluble fibers, especially GG, can reduce emulsification of dietary lipids and lipolysis of TAG in acidic gastric medium in vitro (Pasquier et al. 1996). This may be a mechanism by which soluble fibers can alter lipid assimilation.

In addition, hepatic cholesterol regulates apo B secretion rate by the liver. The lower hepatic cholesterol level in female guinea pigs may result in lower apo B secretion rates compared with males, (Fernandez et al. 1997). In addition, in the case of female guinea pigs, the lower activities of HMG-CoA reductase and ACAT, known to reduce apo B secretion from the hepatocytes (Tanaka et al. 1993), may reduce the secretion of VLDL cholesterol, TAG and apo B by the liver, which explains why male guinea pigs had higher TAG secretion rates than females (Fernandez et al. 1997).

Dietary soluble fiber has an opposite effect on LDL apo B turnover rate compared with dietary cholesterol. Dietary cholesterol reduces LDL receptor-mediated uptake by the liver (Spady and Dietschy 1988), whereas soluble fiber increases LDL receptor-mediated uptake (Fernandez 1995). In this study, specific fiber types altered LDL transport, mainly LDL FCR, in plasma in a different way. In addition there is a gender-associated effect as suggested by our previous reports in males (Fernandez 1995). However, LDL apo B flux was reduced consistently by all types of fiber in both male (Fernandez 1995) and female guinea pigs, suggesting that PE, GG and PSY decrease the conversion rates of VLDL to LDL, possibly by accelerated removal of VLDL by the apo B/E receptor.

From these studies, we conclude that PE, GG and PSY lower plasma total and LDL cholesterol concentrations in female and male guinea pigs. However, different types of fiber appear to have different hypocholesterolemic mechanisms. Gender plays an important role in the response to specific soluble fibers and dietary cholesterol because female guinea pigs are much more susceptible to the effects of a hypercholesterolemic diet. In addition, male and female guinea pigs exhibit different mechanisms in maintaining hepatic cholesterol homeostasis and in regulating plasma lipoprotein metabolism. These findings can help to interpret the relationship among gender, dietary factors and plasma lipid levels associated with coronary heart disease.

	FOOTNOTES

Manuscript received 29 January 1998. Initial reviews completed 21 April 1998. Revision accepted 22 May 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

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