Dietary fiber and related compounds such as oligosaccharides and resistant starches have received considerable attention for their plasma cholesterol-lowering effect. One of these compounds, guar gum (GG),³ a gel-forming galactomannan obtained from Cyamopsis tetragonoloba, received particular attention because of its consistent cholesterol-lowering and glucostatic effects (Gatenby 1990, Todd et al. 1990).

The mechanisms underlying these effects of GG are not fully understood, but one common proposal is that guar gum interferes with the intestinal absorption of steroids, because of its viscosity or its binding properties. GG alters emulsification of dietary fat and lipolysis under conditions prevailing in the upper part of the digestive tract (Pasquier et al. 1996) and delays gastric emptying in dogs (Bueno et al. 1981). In the small intestine, GG also delays lipid dispersion and the rate of absorption of lipolysis end-products, but in rats it is still uncertain whether GG actually affects lipase activity (Ikegami et al. 1990, Poksay and Schneeman 1983). In fact, even if GG does not affect the overall digestibility of glycerides (which remains very high), its presence prolongs the duration of lipid digestion and displaces lipid absorption to a more distal portion of the small intestine (Mazur et al. 1990, Redard et al. 1992). This could alter the structure of triglyceride-rich lipoproteins (TGRLP) released by the digestive tract and their further metabolism, hence their potential atherogenicity (Sethi et al. 1993).

The enterohepatic cycling of steroids, especially bile acids, is considered to be a process particularly prone to interfering effects of sequestrants and polysaccharides such as GG (Stedronsky 1994). Besides inhibition of cholesterol absorption in the upper small intestine, GG could also impair the ileal absorption of bile acids, thus promoting their transfer into the large intestine and their fecal excretion. This spillover of the bile acid pool is liable in turn to elicit an up-regulation of their hepatic synthesis at the expense of the body cholesterol pool. In the rat, the accelerated oxidation of cholesterol to form bile acids may be coupled to an induction of the apolipoprotein B/E (apo B/E) receptor (Mazur et al. 1990), as well as of the microsomal hydroxymethylglutaryl (HMG) CoA reductase activity in the liver (Favier et al. 1995, Moundras et al. 1994). This last response seems to be a good reflection of fiber's capacity to depress plasma cholesterol in rats fed cholesterol-free diets, but it remains to be established whether this mechanism is still operative when a cholesterol-containing diet is fed and HMG CoA reductase activity is repressed.

The aim of the present study, therefore, was to further document the lipid-lowering effect of GG in rats, in the absence or the presence of a moderate level of dietary cholesterol. The present work was more specifically focused on the effects of GG on the enterohepatic cycling of bile acids and on the role of the large intestine in this process.

MATERIALS AND METHODS

Table 1. Composition of the diets [View Table]

In a separate set of anaesthetized rats, a mid-line laparotomy was performed and the bile duct exposed and ligated distally. The bile duct was then catheterized with a PE10 polyethylene tube (Biotrol, Paris, France) and bile was allowed to drain for 5 min before collection into preweighed vials cooled on ice (30 min). Bile volume was determined gravimetrically.

A portion of liver was freezed-clamped and stored at 80°C before extraction of lipids for further determination of triglycerides and cholesterol. In parallel, 2 g of liver were quickly homogenized in 4 mL of an ice-cold buffer (TRIS-HCl 50 mmol/L, sucrose 250 mmol/L, EDTA 50 mmol/L, dithiothreitol 2 mmol/L, leupeptin 1 µmol/L and phenylmethylsulfonyl fluoride 1 µmol/L, pH 7.2), using a loose-fitting Teflon pestle. The homogenate was first centrifuged at 10,000 × g (15 min, 4°C); the resulting supernatant was then centrifuged at 100,000 × g (60 min, 4°C). The pellets were resuspended in 2 mL of the buffer. The centrifugation procedure was repeated and the resulting pellets homogenized in 1 mL of suspension buffer (sucrose 100 mmol/L, KCl 50 mmol/L, KH₂PO₄ 40 mmol/L, EDTA 30 mmol/L, dithiothreitol 1 mmol/L, pH 7.2). The microsomal preparation was stored at 80°C until measurement of enzyme activities. Protein content of the preparation was determined using the Pierce BCA Reagent kit (Interchim, Montluçon, France).

Short-chain fatty acids (SCFA) were measured by gas-liquid chromatography, after ethanolic extraction of plasma samples (Rémésy and Demigné 1974), and on supernatants of cecal contents (40,000 × g, 15 min at 4°C). Bile acids were quantified by an enzymatic procedure, using the reaction catalyzed by 3 -hydroxysteroid dehydrogenase (EC 1.1.1.50; Sigma Chemical, St. Louis, MO). The enzymatic determination was effected either on undiluted plasma or diluted bile (1/100 in normal saline) samples, or after extraction from digestive content samples (small intestine, cecum) or feces by 10 volumes of ethanolic KOH 0.5 mol/L (90 min at 60°C). Neutral steroids were extracted three times with 1 mL hexane from a 100-µL aliquot of the alkaline ethanolic extract after addition of 5-cholestane as an internal standard. The extracts were centrifuged for 5 min at 3000 × g; the solvent was evaporated under a stream of N₂ and the residue dissolved in hexane. Portions (0.5 µL) of this extract were injected into a gas chromatograph (Delsi 330, Paris, France) which was equipped with a 12 m × 0.25 mm (i.d.) fused silica capillary column (BP10, SGE, Villeneuve-St-Georges, France) and a flame-ionization detector. Helium was used as a carrier gas at a pressure of 40 kPa, and the sterols were separated isothermally at 260°C. Sterols were calculated from the peak areas relative to the peak area of the internal standard. Differences in detector response among the various compounds were corrected on the basis of the response factors calculated from a mixture of pure steroids with known molar composition.

Triglycerides (Biotrol) and total cholesterol (BioMérieux, Charbonnières-les-Bains, France) were measured in plasma and lipoprotein fractions by enzymatic procedures. Triglycerides were determined with lipoprotein lipase/glycerokinase/glycerophophosphate oxidase and total cholesterol with cholesterol esterase/cholesterol oxidase; in both cases, H₂O₂ formed was reacted with chloro-4 phenol/amino-4 antipyrine in the presence of peroxydase to form a pink chromogen (500 nm). Liver lipids were extracted and analyzed as described by Mazur et al. (1990). A polyvalent control serum (Biotrol-33 plus, Biotrol) was treated in parallel to samples and served as control of accuracy of results in the analysis of triglycerides and cholesterol.

RESULTS

Table 2. Effects of dietary guar gum in rats fed cholesterol-free or 0.3% cholesterol diets on body weight and cecal fermentations1,2 [View Table]

Table 3. Effects of dietary guar gum in rats fed cholesterol-free or 0.3% cholesterol diets on plasma and liver lipid concentrations1,2 [View Table]

The liver weight was significantly increased by cholesterol supplementation of the diet (+29%), but the liver weight in rats adapted to the GG/cholesterol diet was not significantly different than the value found in controls. In the absence of dietary cholesterol, GG feeding did not affect liver cholesterol or triglyceride concentrations. In rats fed a cholesterol-containing diet, however, there were large increases in the cholesterol (chiefly esterified) and triglyceride concentrations of the liver. GG supplementation drastically reduced this lipid accumulation, to concentrations that were still significantly higher than in control rats (cholesterol) or not significantly different (triglycerides).

Figure 1 illustrates the effects of the diets on plasma lipoprotein cholesterol and triglycerides. Because the rat has very low concentrations of LDL, lipoproteins with a density < 1.040 kg/L (chiefly TGRLP, together with small amounts of LDL) are contrasted with those with a density >1.040 kg/L (HDL fractions). In rats fed cholesterol-free diets, GG lowered cholesterol only in the HDL fraction. In rats fed the cholesterol diet without GG, there was a dramatic rise in the d < 1.040 kg/L lipoprotein cholesterol (especially in TGRLP), but HDL cholesterol was unchanged. When cholesterol was present in the diet, GG had no significant effect on HDL cholesterol, whereas cholesterol in the lower density fraction was strongly decreased (by more than 50%). A triglyceride-lowering effect of GG was observed in the d < 1.040 kg/L fraction in rats fed the cholesterol-free diets. Cholesterol feeding led to a dramatic rise of triglyceride in this fraction, but GG had a potent effect in this case because triglycerides were reduced by more than 50%.

Table 4. Effects of dietary guar gum in rats fed cholesterol-free or 0.3% cholesterol diets on biliary bile acids flux, reabsorption in the intestine and their fecal excretion1,2 [View Table]

The present data indicate that the major part of the bile acid pool is present in the small intestine, but a substantial portion (23-34%) was also found in the cecum. GG led to a marked enlargement of the bile acid pool in the small intestine (+25%) and in the cecum (+98%). Cholesterol feeding was very effective in raising the small intestine (+92%) and the cecal (+144%) pools, but the maximal value was attained in rats fed the GG/cholesterol diet, which exhibited a very high bile acid pool in the cecum (a total of 33 µmol), representing a nearly fourfold increase over the control. The cecal vein-artery difference of bile acids (+0.13 ± 0.02 mmol/L in control rats) was not significantly modified by GG or cholesterol feeding alone (+0.14 ± 0.02 or +0.17 ± 0.01 mmol/L, respectively), but it was significantly increased in rats fed the GG/cholesterol diet (+0.21 ± 0.02 mmol/L). These data, combined with plasma flow measurements, provide a measure of the cecal reabsorption of bile acids; this process represents a substantial part (23% in control rats) of biliary bile acid influx. The cecal absorption was significantly enhanced by dietary GG or cholesterol (to ~10 µmol/h). Absorption of 16.4 µmol/h was observed when both GG and cholesterol were present in the diet; in such conditions, the cecal reabsorption represented 31% of biliary influx. The estimation of the small intestine reabsorption (by difference between biliary influx and cecal absorption plus fecal excretion) shows that the principal reabsorption site of bile acids is the small intestine. This reabsorption was greater in rats fed GG, in parallel to changes in the small intestine pool. In contrast, the small intestine absorption of bile acids was not significantly increased over control levels in cholesterol-fed rats, even though the small intestine pool was doubled in the latter group. Rats fed cholesterol and GG had the highest rate of small intestinal bile acid reabsorption (35.0 µmol/h), but this represented a lower percentage of total bile acid flux (66%) than that of rats fed cholesterol-free diets (>70%).

As shown in Table 5, the biliary flux of cholesterol was not significantly enhanced by cholesterol feeding; furthermore, this flux represented a negligible supply compared with the daily intake of cholesterol by cholesterol-fed rats (186 µmol/d). GG increased bile cholesterol flux, to ~1 µmol/h. Compared with controls, the cecal pool of neutral sterols was 71% greater in rats fed GG, 208% greater in those fed cholesterol and 650% greater (64µmol) in rats fed the cholesterol/GG diet. The daily fecal excretion of sterols in control rats was about 20 µmol/24 h, consisting primarily in coprostanol (73%) and cholesterol (20%); this excretion was doubled by GG. Neutral sterol excretion reached very high values in rats fed a cholesterol diet, and GG further increased this excretion by 50 µmol/24 h (eightfold over the control level). With the addition of cholesterol to the diets, the proportion of cholesterol in fecal sterols increased, whereas coprostanol decreased.

Table 5. Effects of dietary guar gum in rats fed cholesterol-free or 0.3% cholesterol diets on the biliary cholesterol flux and fecal excretion of neutral sterols1,2 [View Table]

Figure 3 shows the respective contribution of bile acids and sterols to the overall elimination of steroids. Sterols made up a major portion of the disposal of cholesterol from the body pool even when cholesterol-free diets were fed. Figure 4 indicates the apparent digestive balance of steroids, namely, the difference between intake (186 µmol/ 24 h in rats fed the cholesterol diets) and excretion. In rats fed cholesterol-free diets, there was a net output of steroids from the body pool. The addition of GG to the cholesterol-free diet approximately doubled the net output of steroids. Rats fed the fiber-free cholesterol diet were the only group with a positive steroid balance: 22% of the daily cholesterol intake was apparently retained. In cholesterol-fed rats, there was a slightly negative balance when GG was present in the diet, suggesting that a substantial endogenous production of cholesterol was still occurring even when the diet contained 0.3% cholesterol.

DISCUSSION

The addition of GG to the diet elicits a marked rise in the viscosity of the digestive contents, which may alter emulsification and hydrolysis of lipids in duodenal medium in vitro (Pasquier et al. 1996). Nevertheless, with a moderate lipid content of the diet, the digestibility of glycerides is likely to remain very high. However, GG may delay lipid digestion, such that absorption occurs in a more distal part of the small intestine (Meyer and Doty 1988), which may affect the size and apolipoprotein composition of TGRLP in rats (Mazur et al. 1990, Redard et al. 1992).

The primary hypothesis concerning the mechanism of the cholesterol-lowering effect of fibers is increased excretion of cholesterol and bile acids. Consistent with this view, GG was found to be effective in enhancing steroid excretion in the present study. Neutral sterols accounted for a major part of steroid excretion, especially in rats fed a cholesterol-containing diet, and it is noteworthy that the excretion of neutral sterols was quite responsive to dietary GG. In rats fed cholesterol-free diets, the biliary secretion of cholesterol extrapolated over 24 h represented a substantial percentage (about 70%) of the daily excretion of sterols. With the addition of GG to the diets, the biliary output of sterols was enhanced, corresponding still to about 60% of sterol excretion. Mucosal cell sloughing also represents a source of endogenous cholesterol in the intestine, and this supply is likely to be increased by dietary GG, which elicits a hypertrophy and an accelerated turnover of the rat intestinal mucosa (Ikegami et al. 1990, Pell et al. 1992). Nevertheless, data obtained in rats fed cholesterol support the view that GG is effective in depressing the absorption of exogenous cholesterol, as previously shown in guinea pigs and rats (Fernandez 1995, Gee et al. 1983), probably by slowing absorption of cholesterol from micelles by mechanisms involving increased resistance to diffusion in the aqueous luminal medium (Gee et al. 1983, Vahouny et al. 1980). This could also be due to the binding of bile acid to fibers or to inhibition of formation of bile acid micelles in the small intestine (Phillips 1986, Vahouny et al. 1980). It must be noted that the fecal loss of bile acids was relatively low because, extrapolating the biliary bile acid fluxes over 24 h, it represented no more than 2% of the biliary bile acid flux in rats fed cholesterol-free diets. In rats fed cholesterol-containing diets, this loss was higher (3.8% in controls and 3.1% in the GG diet group). A stimulatory effect of GG on the secretion of bile acids by the liver has been observed, in keeping with previous data obtained on rat models (Ide et al. 1991, Ikegami et al. 1990). This was the result of a greater bile flow combined with a higher concentration of bile acids in bile. Supplementation of the diet with cholesterol also raised the bile acid flux, with the maximal flux observed when both GG and cholesterol were present in the diet. The digestive tract comprises the largest bile acid pool in rats, and Ide and Horii (1987) have reported that the small intestine and the cecum both contain more than 95% of the pool, located chiefly in the ileum. In the present experiment, GG led to a striking enlargement of the small intestinal bile acid pool, as shown previously (Ebihara and Schneeman 1989), and of the cecal bile acid pool. In parallel, bile acid reabsorption from the small intestine (essentially in the ileum) and the flux of bile acids to the large intestine were enhanced by GG. The small intestinal pool of bile acids was diluted in a larger volume in rats fed GG than in controls, because of the presence of GG itself and the likely presence of greater amounts of endogenous materials (Gee et al. 1996, Johnson et al. 1988). These features, potentially unfavorable to bile acid absorption, could be outweighed by an up-regulation of ileal transport, which occurs in rodents when there is a spillover of the bile acid pool (Lilienau et al. 1993). It has been hypothesized that a dietary load of cholesterol causes an inhibition of bile acid absorption in the ileum (Björkhem et al. 1991), which could explain the concomitant enlargement of the cecal pool in the present study. Accordingly, in rats fed the 0.3% cholesterol diet, the estimated absorption of bile acids in the small intestine was only 18% higher than in rats fed a cholesterol-free diet, even though the corresponding pool was twice as large.

Bile acid transfer into the large intestine plays a major role in the control of bile acid balance. The size of the cecal pool is a reflection of this transfer: it made up 22% of the total intestinal pool in control rats fed the cholesterol-free diet and 32-34% in those fed GG. The effectiveness of bile acid reabsorption from the large intestine is dependent on the solubility of bile acids in the cecum, which is reduced by fibers such as GG (Moundras et al. 1994). GG is readily broken down by the microflora in the cecum and is thus unlikely to play a direct role in bile acid insolubilization; rather, it acts by promoting acidification of the cecal content by the microflora, which tends to insolubilize bile acids. In addition, bacteria may be effective binding sites for bile acids (Gelissen and Eastwood 1995), and they may also synthesize insoluble forms of bile acids (Benson et al. 1993). Nevertheless, bile acid reabsorption from the large intestine corresponded to 23% of the biliary influx in control rats, and this percentage was even higher in rats fed diets containing GG or cholesterol. Thus, the physiological changes elicited by GG in the cecum, such as a greater surface area of exchange or an accelerated blood flow, might outweigh the potential inhibitory effects of GG in the cecal lumen.

In rats fed the cholesterol diet, hypercholesterolemia was caused by a dramatic rise in TGRLP cholesterol, whereas HDL cholesterol was unchanged. This change also elicited a substantial increase in plasma trigycerides, together with an accumulation of triglycerides and cholesterol esters in the liver. Cholesterol ester accumulation is connected to the induction of acylCoA:cholesterol acyltransferase (ACAT), as previously shown (Fernandez 1995, Suckling and Stange 1985). It has been established that cholesterol-enriched diets stimulate hepatic biosynthesis of triglyceride and depress oxidation of fatty acids in rats (Liu et al. 1995). Feeding GG resulted in an almost complete recovery from all these disturbances of lipid metabolism, because hypercholesterolemia and hypertriglyceridemia were practically abolished, and the liver lipid accumulation was drastically reduced. Numerous aspects of the lipid-lowering effects of soluble fibers such as GG occur simultaneously, including reduced availability of dietary cholesterol, changes in plasma apolipoprotein concentrations (especially apo E and apo A-I) (Moundras et al. 1994, Schneeman et al. 1984), accelerated cycling of apo-lipoproteins (Fernandez et al 1995, Mazur et al. 1990) and attenuation of the postprandial rise of glucose and insulin (Morand et al. 1994).

In previous investigations, GG elicited a strong induction of HMG CoA reductase activity in rat liver, in parallel to a cholesterol-lowering effect (Ide et al. 1991, Moundras et al. 1994), but this induction was not observed by Overton et al. (1994). With a cholesterol-free diet, this induction is the result of the GG-mediated diversion of the cholesterol body pool towards fecal steroid excretion. In rats fed diets containing cholesterol and no GG, HMG CoA reductase activity was strongly repressed. When GG was added to the 0.3% cholesterol diets, hepatic cholesterogenesis was probably reactivated, as reported in rats fed pectin or psyllium by Arjmandi et al. (1992). The mechanism of induction of the rate-limiting enzyme of cholesterogenesis by GG is probably connected to a depletion of cholesterol from the liver. In rats fed a cholesterol-free GG diet, there was also an increase in cholesterol 7-hydroxylase activity, suggesting a coordinated up-regulation of HMG CoA reductase and cholesterol 7-hydroxylase (Pandak et al. 1990). However, the situation in cholesterol-fed rats, in which the two enzyme activities changed in the opposite direction, relative to the control rats, suggests a more complex regulation.

The up-regulation of bile acid synthesis by soluble fibers such as GG has been frequently observed in rats (Favier et al. 1995, Ide et al. 1991, Matheson et al. 1995, Overton et al. 1994) but not always in other species such as guinea pig (Fernandez 1995). Because bile acids, especially the nonpolar species, are able to down-regulate cholesterol 7-hydroxylase in rats (Stange et al. 1989), an impaired reabsorption of bile acids should accelerate cholesterol oxidation. In the present experiment, because bile acid absorption remained very effective in rats fed GG, it seems unlikely that the portal concentration of bile acids would be depressed, compared with controls. Nevertheless, cholesterol 7-hydroxylase was strongly induced. The correlation between portal bile acids and the activity of cholesterol 7-hydroxylase has been questioned (Fukushima et al. 1995), and Pandak et al. (1995) have suggested that the down-regulation of cholesterol 7-hydroxylase by bile acids is not an effect exerted by plasma bile acids, but rather by factor(s) released by the intestine when bile acids are present in the lumen.

In conclusion, it appears that a large part of the cholesterol-lowering effect of GG is dependent on its capacity to accelerate neutral and acidic steroid excretion. When cholesterol is present in the diet, GG seems particularly effective in depressing the intestinal absorption of exogenous cholesterol. Basically, GG enhances the intestinal pool of steroids, especially bile acids. Because reabsorption of bile acids is proportional to the size of the intestinal bile acid pool, this process may be substantially enhanced in rats fed GG. Thus, in these rats, there is apparently an enhanced liver secretion and intestinal reabsorption of bile acids, and not merely a diversion of intestinal steroids towards fecal elimination. It is noteworthy that the percentage of the biliary bile acids lost in the feces remained fairly constant with the addition of GG in the diet. Hence, the higher the biliary flux, the greater the fecal elimination of bile acids. In contrast, when cholesterol was added to the diets, the enhanced fecal loss of bile acids corresponded to a less effective reabsorption. In rats fed GG diets, the losses of steroids were compensated for to a certain extent by the induction of liver HMG CoA reductase. The induction of this enzyme took place in spite of an accelerated return of bile acids to the liver; this process could limit the adaptation of cholesterol synthesis and thus contribute to the cholesterol-lowering effect of GG in this animal model.