the binding of bile salts by vegetable fibre

THE BINDING OF BILE SALTS BY V E G E T A B L E FIBRE*

ROBERT R. S E L V E N D R A N

(Norwich)

ABSTRACT

The binding of bile salts by dietary fibre plays an important role in cholesterol metabolism in man. In most of the adsorption studies of bile salts in vitro, the chemical composition of the fibre preparations has not been described, even broadly, nor has the effect of co-precipitated compounds (e.g. proteins) been considered. Hence, it seemed useful to investigate the adsorption of Na cholate (NaC) and Na taurocholate (NaTC) by weU-def'med cell wall material (CWM) from parenchymatous, lignified and cutinised tissues of mature runner bean pods as well as leeks under a variety of experimental conditions. This study was to identify the groups of polymers which are responsible for adsorption. The results showed dramatic differences in the adsorption characteristics of the wall preparations at different pH values. Some of the findings are reported below.

The CWM from the various tissues was prepared by sequentially extracting the wet ball-milled tissues with 1% aq. Na deoxycholate and phenol/acetic acid/water (2:1:1, w/v/v). Experiments with labelled deoxycholate showed that the final preparations contained negligible amounts of adsorbed deoxycholate. Since the amount of residual starch in the preparations was small, no attempt was made to remove it; however, if required, this could be completely removed by extraction with 90% aq. dimethyl sulphoxide. The particle size of the preparations varied from 25-50 p.m. For adsorption studies the foUowing preparations were used: (1) whole-, depeetinated- and delignified- CWM from parenchymatous and lignified tissues of runner beans, together with the H and Na forms; (2) CWM of runner beans at different stages of maturity; (3) CWM of whole- and decutinised-leaves and cutinised tissues of leeks and (4) carboxymethyl cellulose and amberlite resin. The binding of bile salts was measured by a modified isotope-dilution procedure. The neutral sugars from the polysaccharides were determined as alditol acetates by GLC and an estimate of the uronic acid content of the preparations was obtained by a modified carbazole method.

Experiments on the effect of pH on the binding of cholate by the various preparations showed that the adsorption capacity was very much dependent on the pH. The binding increased as the pH decreased. These experiments were complicated by the precipitation cholic acid at a pH value of <4. Nevertheless, an interaction between cholate and CWM persisted in acid solutions. Removal of pectic substances and lignin from the runner bean preparations resulted in a decrease of the adsorption capacity. The results

* Paper read at the 9th International Mutual Congress of Quality Research of the C.I.Q. with the German Society for Quality Research (D.G.Q.) in Reading University, U.K. from 12th to 14th September 1978.

Qual. Plant. - Pl. Fds. hum. Nutr. XXIX, 1-2: 109-133, 1979

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suggested that the adsorption is greatest under conditions in which the ionization of cholic acid and the acidic groups of the cell wall polymers is at its lowest. Following these studies, determinations were made of the adsorption of cholate from an aqueous solution (without buffer) by wall preparations in the H and Na forms. With all the preparations the adsorption was greatest when these were in the H form. Adsorption studies with the preparations from leeks showed that the adsorption capacity of the parenchymatous tissues was considerably more than that of cutinised tissues. Hence, pectic substances in the H form (an not lignin or cutin) are probably the principal binding agents of cholate. The adsorption of Na tauroeholate by the various preparations was independent of pH value. The implications of these results are discussed.

INTRODUCTION

The properties and functions of the polysaccharides in dietary fibre (which includes the structural components of plant cell walls) have been the topic of several investigations. (For some general reviews see: Fifth Annual Marabou Symposium, 1977, Spiller & Shipley, 1977). It has been reported that wheat fibre does not affect plasma lipids in short term experiments in man (Kay & Truswell, 1977a), but some other forms of dietary fibre rich in pectin lower plasma lipids, usually in appreciable amounts (Keys et al. 1961 ; Jenkins et al. 1975; Kay & Truswell, 1977b, Gormtey et aI. 1977). The in vitro experiments of Eastwood and Hamilton (1967)with alcohol-insoluble residues of cereals and vegetables suggested that 'lignin' may be an important bile salt binding polymer. Other workers have observed the ability of detergent extracted residues of plant tissues to bind bile salts in vitro (Kritchevsky & Story, 1974; Eastwood et al. 1976).

Based on these and other related studies, it has been suggested that dietary fibre binds bile salts and other steroids in the intesetine and thus inhibits cholesterol absorption (Balmer & Zilversmit, 1974; Morgan et al. 1974; Eastwood, 1975). In most of the adsorption studies of bile salts in vitro, the chemical composition of the fibre preparations has not been described even in broad terms (Kritchevsky & Story, 1974; Balmer & Zilversmit, 1974; Eastwood et al. 1976). Also, the effect of coprecipitated compounds (e.g. proteins) was not considered. Hence, it seemed useful to investigate the adsorption of Na cholate (NaC) and Na taurocholate (Na TC) by well- difined cell wall material (CWM) from parenchymatous, lignified and cutinised tissues of mature runner bean pods as well as leeks under a variety of experimental conditions. The binding characteristics of carboxymethyl cellulose and ambeflite resin were also studied. A preliminary communicat- ion has been published (Selvendran, 1978).

To obtain a reasonably adequate understanding of the binding process, the following were considered: (1) the nature of the bile salts and the effect of pH on binding; (2) the chemical composition of the adsorbent, especially

110

the presence or absence of polymers which are particularly effective in promoting binding; (3) the particle size of the adsorbent and the rate of binding; (4) the nature of the binding sites; (5) the degree of reversibility of the binding process; and (6) the biological significance of the above points.

MATERIALS AND METHODS

Plant Material.

The parenchymatous and lignified tissues were obtained from mature runner beans grown in experimental plots near the laboratory. Parenchyma was obtained by scraping the inner tisues of the pods (split in half lenght-wise) upto the parchment layer with a spoon. The pods were then steamed and the parchment layers were carefully dissected out and scraped with a blade to remove any loosely held material. They were then blended in an ultra- turrax and the lignified fibres were separated from the debris. The cutinised tissues (epidermal layers) of leeks and the leaves from which the cutinised tissues had been removed were also used.

Preparation of cell wall material ( CWM)

The CWM from the various tissues was prepared by sequentially extracting the wet ball-milled tissues with 1% aq. Na deoxycholate and phenol/acetic acid/ water (2:1:1, w/v/v) as described before (Selvendran, 1975a). Experiments with labelled deoxycholate showed that the t'mal preparations contained negligible amounts of adsorbed deoxycholate. Since the amount of residual starch in the preparations was small, no attempt was made to remove it; however, if required, this could be completely removed by extraction with 90% aq. dimethyl sulphoxide (Ring & Selvendran, 1978). The particle size of the preparations varied from 25-50/xm. The yield of dry CWM from 100g fresh weight of parenchymatous and lignified tissues of mature runner beans, wrtole leeks, decutinised leeks and cutinised tissues of leeks was 1.5, 65, 3.5, 2.8 and 9.4g respectively.

Fractionation of the CWM

The CWM of parenchyma was depectinated by extraction with 0.2M ammonium oxalate (x 3) and the depectinated residue was sequentially extracted with 1 and 4 N KOH containing lOmM Na BH4 to obtain a'-cellulose. The a'-cellulose was associated with some uronic acid containing polymers which showed an incomplete removal of pectic substances. The delignification step was omitted because the tissue is free of lignin.

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The CWM of parchment layers was delignified with warm Na chlorite/ HOAc (Selvendran et al. 1975) and the residue (holocellulose) was washed thoroughly with distilled water. It was then extracted with 1 and 4N KOH containing 10mM Na BH4 for 2 hr each to remove the hemicelluloses. The residue (o~-cellulose) was washed with distilled water and freeze dried. In all these treatments, the sample to solvent ratio was 1:100 (w/v).

Preparations used for binding studies

(1) Whole-, depectinated-, delignified-CWM and a'-cellulose prepared as described above, together with the H- and Na-forms. The H-form was prepared by suspending the CWM (Ig) in N HCL (100rnl) for 2hr and then packing the material in a glass column and washing with distilled water until the washings were free of chloride ions. The Na-form was prepared by suspending the CWM (lg) in 0.5N NaHCO3 (100ml) for 2hr followed by washing with distilled water. A small amount (<5%) of pectic substances was only removed by the above treatment. The preparations were then freeze dried.

(2) CWM of runner bean pods at different stages of maturity (stages 1-8). The average lengths and widths of the pods from stages 1-8 were as follows:- Stage - 1, 7.0 cm and 0.64 cm; stage - 2, 12.5 cm and 0.93 cm; stage - 3, 17.9 cm and 1.2 cm; stage - 4,24 cm and 1.6 cm; stage - 5,25.0 cm and 2.0 cm; stage - 6, 32.0 cm and 2.2 cm; stage - 7, 32.5 cm and 2.2 cm and stage- 8, 32.0 cm and 2.3 cm. Upto stage - 5, there were few phoroglucinol/HC1 staining elements in the transverse sections of the pods, whereas stages - 7 and - 8 contained heavily lignified tissues (parchment layers and strings). The CWM was prepared from the deseeded pods.

(3) CWM of whole- and decutinised leaves and cutinised tissues of leeks. (4) Carboxymethyl cellulose and amberlite resin.

Carbohydrate analysis of the cell wall preparations

The wall preparations were subjected to 2N sulphuric acid and Saeman hydrolysis and the liberated neutral sugars were isolated and analysed as alditol acetates by g.l.c. (Ring & Selvendran, 1978; Selvendran et al. 1978). Hydrolysis with sulphuric acid, unlike Saeman-hydrolysis, does not hydroly- se the ~ (1 >4) links of ceilutose and may be used, therefore, to hydro- lyse preferetially the bulk of the neutral sugars from the non-cellulosic polysaccharides of the wall.

The uronic acid content of the cell wall preparations was determined by an adaptation of the modified carbazole method of Bitter & Muir (1962). Because their method is readily applicable only to uronic acids and soluble

112

uronic acid containing polymers, the latter were first solubilized from the CWM as follows: CWM (10mg) was dispersed in 72% H2 S04 (0.82g) for 3hr at 25~ diluted to 10ml, fffltered through glass fibre triter paper GF/C and an aliquot was taken for uronic acid analysis. Since the neutral sugars gave a small response (5-15% ) to the carbazole reaction, the estimate of uronic acid was corrected for the neutral sugars liberated by Saeman-hydrolysis of the preparation. The relative merits of this procedure are discussed else- where (Selvendran et al. 1978).

Bile salt binding assays

The binding of bile salts was measured by a modification of the isotope dilution procedure of Kritchevsky & Story (1974). Substrate solutions of the bile salts (usually 1-2 mg/ml) in buffer (Giomori, 1955) or distilled water which had tracer quantitites of Na(carboxy-14C) cholate or Na(carbo- nyl A4 C) taurocholate (15 x 10 -4 /ICi/icubation) were used. Na cholate was preferred because cholic acid is only slightly soluble in water. However, in some of the preliminary experiments a saturated solution of chotic acid was used. The standard procedure was as follows: The CWM (10mg) was placed in a screw-topped glass vial ( lcm diam. x 4.5 cm) and the bile salt solution (0.5-1ml) was added. The larger volume was preferred for the parenchyma preparations. The vial was then shaken on a Luckhams rock/roll mixer for 4 hr at 25~ At the end of this period, the contents of the vial were centrifu- ged and a 40#1 aliquot of the supematant was analysed for radioactivity by liquid scintillation spectrometry. The amount of bile salt adsorbed was calculated from the difference between the counts added and that recovered in the supernatant after the incubation period.

RESULTS

Composition of the cell wall preparations from runner beans

The overall composition of the CWM from parenchymatous and lignified tissues of mature runner beans is shown in Table 1. The lignin content of the parchment layers was obtained by subtracting the carbohyarate and protein content of the CWM from its dry weight. The results of the carbohydrate analyses of the various fractions from comparable wall preparations have been reported before (Selvendran 1975b and 1978). The cell wall protein content of the parenchyma and lignLfied preparations was 4.5% and 1.5% respectively. The CWM of parenchymatous tissues contained mainly, pectic substances, cellulose, some alkali-soluble hemicelluloses and hydroxypro- line-rich glycoproteins. Depectination of the CWM resulted in an appreciable

113

lOSS of its uronic acid content; the oxalate-soluble polymers accounted for

25-30% of the dry weight of the CWM. The CWM of parchment layers contained mainly lignin, cellulose and xylans. From the holocellulose, xylans containing about 4% uronic acid residues could be solubilised with alkali. Unlike the a'-cellulose from parenchyma, the a-cellulose from parch-

ment layers gave mainly glucose (>97%) on Saeman-hydrolysis, showing that it is virtually free of other cell wall polymers.

Tabel 1 Percentage composition (w/w) of the CWM of parenchymatous and lignified tissues of runner beans

Parenchyma Parchment layers Pectic substances 35-40 Lignin 35-40 Hemicelluloses 20-25 Hemicelluloses 25-30

(Xylans) Glycoproteins* 10-15 Glycoproteins* ~ 5 a, Cellulose 30-35 a-Cellulose 35-40

* Note that the glycoprotein content is different from the protein content.

Composition of the cell wall preparations from leeks

The carbohydrate composition of the CWM from decutinised tissues (which contain mainly chlorenchyma cells) and cutinised tissues of leeks is shown in Table 2. The cell wall protein content of the decutinised and cutinised tissues was 9.2% and 1.2% respectively. From these results, the following overall composition of the cell wall preparations could be inferred. Decutini-

Table 2. Comparison of the carbohydrate composition of CWM from decutinised and cutinised tissues of leeks (results expressed as //g sugar/mg dry preparation and are from single analysis)

CWM of decutinised tissues CWM of cutinised tissues H2SO4- Saeman- H2SO4- Saeman-

hydrolysis hydrolysis hydrolysis hydrolysis

Rhamnose 27.8 25.8 37.6 16.6 Arabinose 39.0 36.0 59.1 55.1 Xyloso 40.0 41.6 25.6 24.2 Mannose 10.1 22.4 8.4 34.6 Galactose 157.2 154.0 71.4 65.6 Glucose 12.5 303.0 39.8 195.5 Uronic acid* - 297.5 - 37.4

* The uronic acid content of the preparations was determined by a modified carbazole method.

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sed tissues: cellulose, 313%; polyuronic acid (polygalacturonic?) 30.6% ; hemicelluloses, 28.9%; cutinised tissues: cutin (by difference in weights)l 55.5% ; hemi-celluloses, 23.6% ; cellulose, 15.6% ; polyuronJc acid, 3.7% In the above values, the 'hemicelluloses' include the contribution from the neutral sugar fraction o f pectic substances.

4 0 0

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13)

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0

E "" 2 0 0

t - O

. i

o

<

100

I / ' ~ I / ' ~ I ,4~ I

2 4 6 8

Time hours

Fig. 1. The rate of binding of Na cholate from aqueous solutions (without buffer) by cell wall preparations from parenchymatous tissues of runner beans.

In this and the following figures, all the values are means of duplicate determinations. Key: *, whole-CWM (H-form)-: &, Depectinated-CWM (H-form); O, Whole-CWM (Na- form).

115

Rate of binding of'NaC by CWM

The rate of binding of NaC from aq. solutions (without buffer) by the cell waU preparations from parenchymatous and lignified tissues of runner bean pods are shown in Figs. 1 and 2. Comparable binding rates were obtained when buffer solutions were used instead of distilled water. Most of the

40

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Fig. 2. The rate of binding of Na cholate from aqueous solutions (without buffers) by CWM from parchment layers, Key: e, Parehment-CWM (H-form); O, Parchment-CWM (Na-form).

116

binding (~95%) occurred in the first 4hr. When the binding was allowed to proceed for periods longer than 4hr, a further increase in binding occurred. However, this increase amounted to only a few percent of that adsorbed in the first 4hr and may be associated with a slow binding into the interstices of the wall matrix. Therefore, 4hr was used for the incubation period in the following experiments. It is of interest to note that the CWM of parenchyma (Na-form) has neglignible binding capacity. The significance of these results will be discussed later.

Effect of particle size of lignified tissues on binding capacity

Table 3 shows the effect of the particle size on binding capacity. From the results, it is clear that the particle size of the CWM of lignified tissues has little effect on the binding capacity. The preparation with particle size ranging from 1-1.5/am length and 1-1.5/1m width was obtained by dry bali- milling the freeze-dried CWM. It is possible that a certain amount of degradation of the cell-wall polymers occurs on dry baU-milling. However, suitable checks carried out with known polysaccharides and proteins showed that the wet-bailing procedure (for 15-20hr) used for the preparation of the CWM did not result in detectable degradation of the polymers. Wet ball-milling, however, ensured (almost) complete disruption of the various types of cells, allowing the solvents to penetrate the sample fully and dissolve the intracellular compounds.

Table 3. Effect of particle size of Ugnified tissues (parchment layers) on Na cholate binding at pH 5

Fibre Type Particle size in/.a-n n moles adsorbed/ mg preparation

Length Width Coarse 1000-1500 120-350 26.1-+2.3 Fine 250- 500 15- 40 28.2+2.4 Ball-milled 10- 15 10- 10.5 34.1-+2.6

Values are means of 5 determinations + SD

Effect of pH on binding

The effect of pH on the binding of NaC and NaTC by the cell wall preparations from both parenchymatous and lignified tissues is shown in Fig. 3. The pH was checked both before and after the period of incubation and the average value was used. For convenience the binding of NaC and NaTC will be considered separately.

117

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E

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150

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1

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I

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3 4 5 6 7

pH Fig. 3. Effect of pH on the binding of Na cholate (NaC) and Na taurocholate (NaTC) by cell wall preparations from parenchymatous and lignified tissues of mature runner beans, Key: 0, Parenchyma-CWM/NaC; I, Parenchyma-CWM/NaTC; o, Parchment- CWM/NaC; O, Parchment holocellulose/NaC; A, Parchment ~-cellulose/NaC; A, Parchment-CWM/NaTC.

(a) Binding of NaC

Experiments on the effect of pH on the binding of cholate by the various preparations showed that the binding capacity was very much dependent on the pH. It can be seen that both the affinity (initial slope) and binding capacity, increased with decreasing pH. These experiments were complicated by the precipitation of cholic acid at pH <4. Nevertheless, an interaction between cholate and CWM persisted in acid solutions. At pH 4, the binding of cholate by CWM of parenchyma (190 nmoles/mg) was considerably more than that of parchment layers (70nmoles/mg). About 30-40% of the adsorbed cholate could be desorbed by changing the pH of the medium from 4 to 8. However, absolute alcohol desorbed ~ 90% of the

118

bound cholate. Removal of pectic substances, lignin and xylans, from the respective preparations, resulted in a decrease of the binding capacity.

(b) Binding of NaTC

To clarify the effect of increased acidity and solubility of the bile salt on the binding mechanism, the adsorption of NaTC was studied. Taurocholic acid differs from cholic acid in having a sulphonic acid group which confers on it higher acidity and solubility. The binding of NaTC by CWM of both parenchymatous and lignified tissues was independent of pH. The pK of taurocholic acid is 1.5 and it would therefore, be in the fully ionized form at pH values >4 and hence its binding would be independent of pH within the pH range studied.

The increased solubility of the salt also could contribute to the effect observed.

Effect of the H-and Na-forms of the CWM on NaC binding

Following these studies, determinations were made of the binding of cholate from an aq. solution (without buffer) by wall preparations in the H and Na forms. The time course of the binding results are shown in Fig. 1 and 2. With all the preparations it was clear that the binding was greatest when these were in the H form; that is to say under conditions in which the ionization of the acidic groups of the wall polymers is very much reduced. This is also borne out by the fact that the Na forms of the CWM and depectinated CWM of parenchyma had negligible binding capacity. This decrease in binding capacity could be due to the increased repulsion between the cholate ions and the carboxylate groups of the CWM. The results also suggest that the extent of binding would be dependent upon the carboxyl group density of the CWM.

The results with lignified tissues shows that the H-form of the preparation binds much less cholate compared with the corresponding preparation from parenchyma. This would preclude the lignin polymer from being a major binding site. However, the Na-form also binds some cholate, showing that hydrophobic interactions (probably with lignin) contribute to the binding process.

Comparison of the binding capacities of CWM and some weak cation exchange resins

Because the extent of binding may be dependent upon the carboxyl group density of the adsorbent, a comparative study was made of the binding

119

capacities of CWM of runner beans and some weak cation exchange resins (all in H-form).

From the titration curves of the CWM, carboxymethyl cellulose (CMC) and amberlite resin, an estimate of the number of titratable carboxyl groups/mg material was obtained. The results of the titration and binding experiments are summarized in Table 4. The titratable acidic groups in the CW'M of parchment layers was very smaU. From the results it is clear that the CWM of parenchyma has the highest binding capacity and that there are other factors besides the number of free carboxyl groups (e.g. conformation of the macromolecules) which determine the ability of a polymer to bind NaC.

Table 4 Comparison of the binding capacities of CWM of runner beans and some weak cation exchange resins*

Adsorbent (Ad.) Titratable /.t eqts. acid/ //moles NaC No. of -COOII groups 100mg Ad. adsorbed/100 gps of Ad./NaC

mg Ad.

CWM - Parenchyma mainly 320 33 9.7 -COOII

CWM - Parchment layers -COOII ** 3.5 - or phenolic-OH(?)

Carboxymethyl cellulose -COOH 230 4.6 50 Amberlite resin -COOH 1509 17 88.8

* All the polymer preparations were in the H-form ** The acidity of the CWM was too small that an estimate could not be obtained.

Binding of NaC by CWM of runner bean pods at different stages of maturity

A set of experiments were designed to study the effect of the maturity of the pods on the binding of NaC. The results of experiments with CWM of pods at different stages of maturity at pH values 5 and 6 are shown in Fig. 4. The protein content of the preparations varied from 4 to 5.5% of the dry weight of the CWM. It is clear that while pH has a definite effect on binding, the maturity of the pod has negligible effect. This is presumably due to the fact that the bulk of the binding is by CWM of parenchymatous tissues (parenchyma and chlorenchyma) of the pods.

Binding of NaC by CWM of leeks

At cholate concentration of lmg/ml, the binding capacities of the CWM of

120

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100

50

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pH6

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o

I I I I L I I I

1 2 3 4 5 6 7 8

Stage of maturity

Fig. 4. Binding of NaC to CWM of runner beans at different stages of maturity.

whole leeks, decutinised leeks and cutinised tissues were 47-+5.1, 53.14"5.8 and 14.44"1.7 nmoles/mg preparation respectively at pH 5. This constitutes evidence that cutin has very little NaC binding characteristics. Cutin is indigestible, but, unlike other cell wall polymers, is not uniformly distribut- ed throughout plant tissues but confined to the outer surfaces.

Effect o f NaC concentration on binding

The CWM from parenchymatous and lignified tissues of runner beans (in the H-form) were exposed to increasing concentrations of NaC (1-16 mg/ml) and the adsorption results are shown in Fig. 5. NaC at concentrations>3 mg] ml tended to precipitate from solutions of pH 5 therefore higher concentrations were not tested at this pH. The graphs at pH 7 show characteristic forms. With CWM of parenchyma, below 3 mg/ml, the binding is nearly independent of concentration, but at this concentration the slope of the curve changes abruptly. This concentration probably corresponds to the onset of increased association of the 'hydrocarbon face' of the cholate ions adsorbed onto the CWM with those in solution. This association phenomenon is comparable with the formation of micelles in bulk solution (and has been called hemimicelle formation). At concentrations >14mg/ml, there is a marked increase in binding, probably indicating a change in orientation of

121

%)

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ook ? /

400 ~

300 -

200

100

r o.0/ 0.._91 I I I

2 6 10 14

Conc Na cholate mg / ml Fig. 5. The extent of binding of NaC to CWM of runner beans as a function of NaC concentrg_t.ion, Key: A, Parenchyrna-CWM, pH 7; e, Parenchyma-CWM, p H 5; O, Parch- ment-CWM pH 7.

the adsorbed molecules (from parallel to perpendicular?). The results with lignified tissues is somewhat comparable with parenchy-

ma, except that the binding capacity is considerably less and the initial increase in binding is followed by a decrease in the range 7-10mg/ml. This effect is presumably due to the fact that anions (citrate and phosphate) from the buffer are preferentially adsorbed.

DISCUSSION

The main aim of the present investigation was to obtain quantitative results

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on the binding of bile salts by CWM of parenchymatous, lignified and cutinised tissues of vegetables after various treatments. A knowledge of the mechanism of the bile salt/acid - CWM interaction may prove relevant to the ultimate understanding of the possible importance of dietary fibre. Because of the difficulties in estimating accurately small amounts of bile salts, by the colorimetric method, an isotope dilution method was used. The rate of binding was slow and the amount of 'bile salt' adsorbed was some- times increasing with time even after 4 hr of vigorous agitation of solution and adsorbent. This may be due to the slow diffusion of the bile salt into the cell wall matrix before equilibrium is attained. Reducing the particle size of the CWM of lignified tissues had little effect on the binding capacity, but permitted a more rapid saturation of the wall's binding sites. In a series of experiments in which the various groups of polysaccharides were removed from the wall material, the importance of the acidic groups of the pectic substances and to a lesser extent that of lignin and xylans in bile salt binding was shown.

The most interesting result was the dependence of the binding of NaC on the pH of the media, which suggested the possible importance of the acidic groups in the CWM for binding. The binding capacity of the CWM increased with decreasing pH. The decrease in binding with increase in pH is coinci- dent with the titration curve of the CWM (H-form) in the carboxyl ionization pH range. In the pH range 5.5 to 4.0, there was a marked change in the binding characteristics of the preparations (the pK values of cholic acid and galacturonic acids are 5.0 and 4.2 respectively). These findings suggested that the binding is greatest between the unionized species of both cholic acid and the acidic groups of the cell wall polymers (e.g. - COOH groups of pectic acid). The degree of binding of cholic acid is probably dependent on its degree of ionization and solubility. The marked decrease in binding at pI-E>6 is probably caused by the electrostatic repulsion of the anionic cholate by the ionised carboxyl groups of the CWM and the increased solubility of the salt at higher pH. It is probable that many of the anomalous results obtained by workers in this field could be due to the use of various wall preparations, with differing amounts of carboxyl and carboxylate groups.

A comparison of the binding properties of NaTC and NaC showed that the binding of the former was independent of pH. This is presumably due to the increased solubility of NaTC and the very low pK of the acid, which enables it to remain in the ionized form throughout the range of pH studied.

The results of experiments on the binding of NaC by CWM and depectinated CWM in the H- and Na-forms confirmed the above picture. With CWM of parenchyma, the principal component which binds the bile salt is pectic substances (probably pectic acid) and it could be inferred from the results

123

that Na-Pectate has negligible adsorption capacity. It seems likely that the carboxyl and hydroxyl groups of the bile salt/acid forms hydrogen bonds with similar groups of pectic acid. The relative amount of NaC adsorbed by CWM of lignified tissues (H-forms) is very much lower than the corresponding preparation from parenchyma. These results suggest that lignin is not a major binding site. The observation that the Na-form of the CWM of lignified tissues binds more cholate compared with the corresponding preparation from parenchyma shows that hydrophobic interactions (probably with lignin) make a contribution to binding. The nature o f the association is probably complex in which the 'hydrophobic face' of the bile salt is also involved in bonding. Interactions with hemicelluloses, mainly xylans, are also involved but they are relatively minor. The results of binding experiments with CWM of beans at different stages of maturity also show that the wall polymers of parenchymatous tissues are the major binding sites of NaC. Although bile salts can bind to proteins and can extract intracellular proteins from plant tissues, the contribution o f wall proteins to binding must be small, because of the low protein content of the preparations. Further, the wall proteins are fully embedded in the wall matrix and are not readily available for binding.

MECHANISM OF NaC BINDING BY CWM

Primary and secondary NaC micelles

NaC unlike the usual aliphat.ic detergents which possess a clear-cut polarity between hydrophobic and hydrophilic parts, prossesses a rigid ring structure, one side of which is spiked with hydroxyl groups and the other side with methyl groups. Further, one end contains a short hydrocarbon chain which ends in a carboxyl group. NaC can therefore be viewed as a rigid ring structure (having a steroid nucleus) with hydrophilic and hydrophobic sides to which is attached a hydrophilic tail. In the small primary micelle, NaC molecules, like detergent molecules, probably associate by hydrogen bonding and hydrophobic interactions. The bonding o f the cholate molecules, however, is back to back rather than chain to chain as with detergents. Secondary cholate micelles are aggregates of primary micelles (see Fig. 6).

Association of NaC with CWM

It would appear from structural considerations that NaC is adsorbed on the CWM in the form of layers of close-packed sausage-like molecules lying flat against the substrate. From molecular models one cholate ion or molecule

124

D

Na cholate

longitudinal section

Micelles

cross section

Aggregat ion number 2 - - 8

Fig. 6. Longitudinal and cross sections of primary and secondary Na cholate miceUes (from Small, 1968).

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could be associated with five galacturonic acid residues of pectic acid. The observation that one molecule of cholate associates with approximately ten galacturonic acid residues (see Table 4) instead of five suggests that no t all of the latter are equally accessible to the cholate. The difference can easily be explained on the assumption that some of the galacturonic acid residues are hindered by bulky side groups and are not readily accessible to the cholate micelles or they can be regarded as bonded in an organised structure.

The structure of the cholate-pectic acid aggregate as deduced f rom the above discussion is shown in fig. 7a. In this proposed arrangement, which must be considered topographical, the cholate/cholic acid molecules, adsorb at the pectic acid - water interphase and H-bond through hydroxyl and carboxyl groups of both molecules. The charged group extends into the aq. phase and stabilizes the system. It is probable that one or more water molecules participate in this H-bonding, bridging the OH groups. On increasing the cholate concentration, the molecules may be arranged in pairs with their hydrophobic backs next to each other. Still further increase in cholate concentration may lead to the formation of aggregates comparable with secondary cholate micelles. The size that such aggregates can attain must, however, be limited. When the aggregation proceeds further, a new arrangement must sooner or later result in which part of the ions turn so that their hydrophilic groups approach each other as in the termolecular Fdms. Ion-dipole interaction may play a role in keeping these anions together. At concentrations greater than 14 mg/ml, it is probable that a change in orientation of the adsorbed molecules (from parallel to more or less perpendicular to the wall surface) takes place. With lignified tissues, the mechanism for cholate-CWM interaction is probably more complex in which the 'hydrophobic" face' of the bile salt is also involved in bonding (Fig. 7b).

The biological significance of the results

Before discussing the possible implications of bile salt - dietary fibre interactions in the human digestive tract, it is useful to summarize some relevant aspects of bile salt/acid metabolism in mammals.

Metabolism of bile salts/acids

The main bile acids in the bile of most mammalian species are cholic acid, chenedeoxycholic acid and deoxycholic acid. These acids are present in the bile as conjugates with taurine and glycine. Since both taurine and glycine conjugates have relatively low pK (approximately 1.5 and 3.5 respectively) and the intestinal pH is about 6.2, the conjugates are completely in the ionic

126

Na iho late

)

Pect

Hydrogen bonding

A B

Fig. 7a. Possible orientations of Na cholate molecules at pectic acid water interphase; as noted, the bile salt molecules may be arranged in pairs with their hydrophobic backs adjacent. How the water molecules are orientated in the aggregate is not certain. Fig. 7b. Possible orientations (A, B) of Na cholate molecules at lignified tissue-water interphase. The adsorbed bile salt molecules may be either ionized or non-ionized.

form. The pK of the unconjugated bile acids is approximately 6.4 and hence they may occur in the lumen of the ileum in both the ionized and unionized forms. The ratio of the taurine to glycine conjugated bile acids varies with the species, age, diet, hormones and liver disease. Bile salts are excreted with the bile into the intestine, reabsorbed in the ileum and again excreted with the bile. During this enterohepatic circulation of bile salts/acids, part of the bile acids is not reabsorbed and is excreted with the faeces. Non-absor- bable components of the diet may influence the absorption of bile salts. (Norman 1964; Eastwood & Boyd, 1967; Eastwood & Hamilton 1968). In the intestine, the bile salts are deconjugated and are subjected to the action of micro-organisms, which results in structural modifications of the bile acids. These reactions begin in the lower part of the ileum and continue through the large intestine. Many of these metabolites appear to be poorly reabsorbed. The bile acids excreted with the faeces consist of a complex

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mixture of microbially formed metabolites. The bile acids reabsorbed from the intestine are conjugated with taurine or glycine in the liver.

In healthy subjects on a normal diet the half-life of cholic acid is 2-3 days and the amount of daily synthesis of cholic acid is about 350rag. The excretion of bile acids in faeces has been measured by several investigators and the values reported vary within a wide range, from less than 100 mg to over lO00mg per day. The values for neutral steroid excretion obtained by different group of workers were 500-700 mg/day. The differences between the reported values can be ascribed, in part, to differences in dietary fibre intake.

The concentration of bile acids in human serum was found by Sandberg et al. (1965) to be 0.03 - 0.23 mg/10Oml of serum. Deoxycholie acid, chenodeoxycholic acid and cholic acid were the major bile acids and they were present mainly as conjugates.

(The above summary was extracted from the article on 'Formation and metabolism of bile acids' by Danielsson & Einarsson, 1969).

Bile salt - dietary f ibre interactions

Several workers have shown that diet rich in pectin lowers serum cholesterol in man (suggesting that pectin is potentially a valuable component o f the diet). The results of our investigation show how the bile salt binding properties of vegetable fibre (at acid pH) could explain this phenomenon. Although the secretions from the bile duct would be alkaline, they come into contact with partially digested food from the stomach at acid pH ("2-3). Thus appreciable binding of the bile salts to the fibre in the food would take place. The bound bile salts/acid might be accessible for absorption only to a limited extent. The low solubility of some bile acids may also influence the extent of absorption. Further, only a small proportion (~30%) of the bound acid would be desorbed when the pH increases to 7 in the lower part of the small intestine.

The amount of NaTC and NaC bound by CWM ofparenchyma at pH 5 is 48 and 52 nmoles/mg respectively. From these figures it could be computed that the amount of NaTC and NaC bound by 10g of parenchyma fibre (which would be about the average daily intake of vegetable fibre/head) would be 266 and 225 mg respectively. These figures are of the same order of magnitude as the amount of bile acids excreted in the faeces per day by an adult. In this connection it is of interest to note that some of the dietary fibre constituents (particularly pectic substances) are appreciably degraded (by micro-organisms) in the large intestine (Cummings et al. 1978). This would result in the release of a proportion of the bound bile salts/acids. However, only a small proportion of the released acids (which may be

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modified by micro-organisms to secondary bile acids) would be reabsorbed in the large intestine and the bulk of the acids would be excreted. It is also

conceivable that the dietary effects are mediated by changes in the intestinal microflora as well as by changes in the rate of passage of content through the intestinal tract. Since the bile salts excreted must be replaced by additional synthesis from cholesterol, this would result in a compounded

drain of body cholesterol pools. Thus the enhanced elimination of bile salts by diet rich in pectic substances is a means of removing hepatic cholesterol, resulting in lower blood cholesterol levels.

More work on the properties of dietary fibre, and different preparations used for chnical feeding trials, have to be done to obtain a full knowledge of the bile salt dietary fibre interactions. It is also important to obtain quantitative characteristics of different fibre preparations for practical purpose. Such investigations are in progress.

Acknowledgement

I would like to dedicate this paper to the memory of my beloved mother (Mrs. P. S. Rasiah) who taught me to think critically and objectively.

ZUSAMMENFASSUNG

Die Bindung yon Salzen des Gallensaftes dutch Ballaststoffe spielt eine wichtige Rolle im Cholesterol-Metabolisrnus des Menschen. In den meisten der Adsorbtions-Studien der Salze des Gallensaftes in vitro wurde weder die chemische Zusammensetzung der Fiber-Pr/ipaxate beschrieben - eventueU ganz allgernein - noch betrachtete man die Wirkung gleichzeitig ausgefbalter Substanzen, wie z.B. der Proteine. Daher erschien es efforderlich, die Adsorbtion yon Natriumcholat (NaC) und Natriumtaurocholat (NaCT) dutch wohldefmiertes Zellwand-Material (CWIVl) yon parenchymatischem, lignifizier- tern und kutinisiertern Gewebe reifer Samen der Feuerbohne (Phaseolus coccineus) sowie yon Porree (Allium ampeloprasum vat. porrum) unter variierten experirnenteUen Bedingungen zu untersuchen. Dies bezog sich auf die Identif'lzierung der polymeren Gruppen, die ffir die Adsorbtion verantwortlich shad. Die Ergebnisse weisen entschei- dende Unterschiede in den Adsorbtionsg2harakteristika der Zellwand-Pr/iparate bei verschiedenen pH-Werten anf. Uber einige der Ergebnisse sou irn folgenden berichtet werden.

Das CWM der verschiedenen Gewebe wurde gewonnen dutch rnehrmals durchge- fiihrte Extraktion, indem das Material rnit 1%igern w/i~rigen Natriurn-Deoxycholat und mit Phenol/Essigs/iure/Wasser (2:1:1, G/V/V) feucht in der Kugelmtihle behandelt worden war. Versuche mit rnarkiertern Deoxycholast zeigten, dat~ die Endpr~parate zu vernaehl/issigende Mengen yon adsorbiertem Deoxycholat enthielten.

Adsorbtions-Studien rnit den Porree-Pr~paraten zeigten, dag die Adsorbtions-Kapazi- t/it der p~enehymatischen Gewebe betr~chtlich h6her war als das der kutinisierten. Hieraus folgt, dat~ Pektine ha der H-Form - und nicht Lignin oder Kutin - wahrscheha- lich die haupts/ichlichen bindenden Agentien yon Cholaten shad. Die Adsorbtion yon

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Natrium-Taurocholat war bei den verschiedenen Prgparaten vom pH-Wert unabh~/ngig. Es werden die Folgerungen aus diesen Ergebnissen besprochen.

SUMMARY

The binding of bile salts by dietary fibre plays an important role in cholesterol metabolism in man. In most of the adsorption studies of bile salts in vitro, the chemical composition of the fibre preparations has not been described, even broadly, nor has the effect of co-precipitated compounds (e.g. proteins) been considered. Hence, it seemed useful to investigate the adsorption of Na cholate (NaC) and Na taurocholate (NaTC) by well-defined cell wall material (CWM) from parenchymatous, lignified and cutinised tissues of mature runner bean pods as well as leeks under a variety o f experimental conditions. This study was to identify the groups of polymers which are responsible for adsorption. The results showed dramatic differences in the adsorption characteristics of the wall preparations at different pH values. Some of the findings are reported below.

The CWM from the various tissues was prepared by sequentially extracting the wet ball-milled tissues with 1% acq. Na deoxycholate and phenol/acetic acid/water (2:1:1, w/v/v). Experiments with labelled deoxycholate showed that the final preparations contained negligible amounts of adsorbed deoxycholate. Since the amount of residual starch in the preparations was small, no attempt was made to remove it; however, if required, this could be completely removed by extraction with 90% aq. dirnethyl sulphoxide. The particle size of the preparations varied from 25-50 b~rn. For adsorption studies the following preparations were used: (1) whole-, depectinated- and delignified- CWM from parenchymatous and lignified tissues of runner beans, together with the H and Na forms; (2) CWM of runner beans at different stages of maturity; (3) CWM of whole- and decutinisedqeaves and cutinised tissues of leeks and (4) carboxyrnethyl cellulose and amberlite resin. The binding of bile salts was measured by a modified isotopedilution procedure. The neutral sugars from the polysaccharides were determined as alditol acetates by GLC and an estimate of the uronic acid content of the preparations was obtained by a modified carbazole method.

Experiments on the effect of pH on the binding of cholate by the variouspreparations showed that the adsorption capacity was very much dependent on the pH. The binding increased as the pH decreased. These experiments were complicated by the precipitation of cholic acid at a pH value of ~4. Nevertheless, an interaction between cholate and CWM persisted in acid solutions. Removal of pectic substances and lignin from the runner bean preparations resulted in a decrease of the adsorption capacity. The results suggested that the adsorption is greatst under conditions in which the ionization of cholic acid and the acidic groups of the cell wall polymers is at its lowest. Following these studies, determinations were made of the adsorption of cholate from an aqueous solution (without buffer) by wall preparations in the H and Na forms. With all the preparations the adsorption was greatest when these were in the H form. Adsorption studies with the preparations from leeks showed that the adsorption capacity of the parenchymatous tissues was considerably more than that of cutinised tissues. Hence, pectic substances in the H form (and not lignin or cutin) are probably the principal binding agents of cholate. The adsorption of Na taurocholate by the various preparations was independent of pH value. The implications of these results are discussed.

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R E F E R E N C E S

Balmer, J. & Zilversmit, D.B. (1974). Effects of dietary roughage on cholesterol absorption, cholesterol turnover and steroid excretion in the rat. J. Nutr. 104:1319- 1328.

Bitter, T. & Muir, H. M. (1962) A modified uronic acid carbazole reaction. Anal Biochem 4:330-334.

Cummings, J. H., Southgate, D. A. T., Branch, W. Wiggins, H.S., Houston, H., Jenkins, D.J.A. and Hill, M.J. (1978) The digestion of dietary pectin in the human gut and its relation to calcium adsorption and large bowel function. Brit. J. Nutr. (in press).

Danielsson, H. & Einarsson, K. (1969). Formation and metabolism of bile acids. In: 'The biological basis of medicine' (ed. E.E. Bittar & N. Bittar) pp. 279-315. London, New York: Academic Press

Eastwood, M.A. & Boyd, G.S. (1967) The distribution of bile salts along the small intestine of rats. Biochem. Biophys. Acta 137,393-396.

Eastwood, M.A. & Hamilton, D. (1968). Studies on the adsorption of bile salts to non- adsorbed components of diet. Biochim. Biphys. Acta 152,165-1 73.

Eastwood, M.A. (1975). Vegetable dietary fibre - potent pith. Royal Society o f Health Journal, August issue.

Eastwood, M.A., Anderson, R., Mitchell, W.D., Robertson, J. & Pocock, S. (1976). A method to measure the adsorption of bile salts to vegetable fibre of differing water holding capacity. J. Nutr. 106, 1429-1432.

Fifth Annual Marabou Symposium on 'Food and Fibre' In: Nutrition Reviews (1977) vol. 35.

Gomori, G. (1955) Preparation of buffers for use in enzyme studies. In: 'Methods in Enzymology' (ed. $2 . Colowick & N.O. Kaplan) vol. 1, pp. 138-146 New York: Academic Press.

Gormley, T.R., Kevany, J., Egan, J.P. & McFarlane, R. (1977) Effect of apples on serum cholesterol levels in humans, lr. J. FcL Sci. TechnoL 1,117-128.

Jenkins, D.J.A., Leeds, A.R., Newton, C. & Cummings, J.H. (1975) Effect of pectin, guax gum and wheat fibre on serum cholesterol. Lancet 1, 1116-1117.

Kay, R.M. & Truswell, A.S. (1977a). The effect of wheat fibre on plasma lipids and faecal steroid excretion in man. Brit. J. Nutr. 37: 227-235.

Kay, R.M. & Truswell, A.S. (1977b) Effect of citrus pectin on blood lipids and faecal steroid excretion in man.Am. J. Clin. Nutr. 30:1 71-1 75.

Keys, A., Grande, F. & Anderson, J.T. (1961) Fibre and pectin in the diet and serum cholesterol concentration in man. Proc. Soc. Exp. Biol. Med. 106:555-558.

Kritchevsky, D. & Story, J.A. (1974) Binding of bile salts in vitro by non-nutritive fibre. J. Nutr. 104:458-462.

Morgan, B., Heald, M., Atkin, S.D., Green, J. & Chain, E.B. (1974) Dietary fibre and sterol metabolism in the rat. Brit. J. Nutr. 32:447-455.

Norman, A. (1964) Faecal excretion products of cholic acid in man. Brit. Z Nutr. 18:1 73-186.

Ring, S.G. & Selvendran, R.R. (1978) Purification and methylation analysis of cell wall material from solanum tuberosum. Phytochemistry 17: 745-752.

Sandberg, D.H., Sjovall, J., Sjovall, K & Turner, D.A. (1965) Measurement of human serum bile acids by gas-liquid chromatography. J. Lipid Res. 6:182-192.

Selvendran, R.R. (1975a) Analysis of cell wall material from plant tissues: extraction and purification. Phytochemistry 14:1011-1017.

Selvendran, R.R. (1975b) Cell wall glycoproteins and polysaccharides of paxenchyma

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of Phascolus coccineus. Phytochemistry 14:2175-2180. Selvendran, R.R., Davies, A.M.C. & Tidder, E. (1975) Cell wall glycoproteins and

polysaccharides of mature runner beans. Phytoehemistry 14:2169-2174. Selvendran, R.R. (1978) Bile salt binding sites in vegetable fibre. Chemistry and

lnclustry, June issue, pp. 428-430. Selvendran, R.R., March, J.F. & Ring S.G. (1978) Determination of aldoses and

aronic acid content of vegetable fibre. Anal. Biochem. (in press) Small, D.M. (1968) Size and structure of bite salt micelles. In: 'Molecular associat-

ion in biological and related systems' - Advances in chemistry series 84 (ed. R.F. Gould) pp. 31-52. Washington, D.C.: American Chemical Society.

DISCUSSION

Dr. Hellendoorn (Zeist/The Netherlands):

Your slides on the effect of wet ball-milling the tissues, which results in complete disruption of cell structure were quite impressive. The pre-treatment of the sample has certainly to be introduced, replacing disintegration in a blender in the method for the determination of indigestible residue (dietary fibre) content of foods (J. Sci. Food Agric. 1975, 26,1461-1468.

Dr. Selvendran: (Norwieh /G.B.) :

Yes, I agree that tissues have to be wet ball-milled for complete disruption of cell structure. However, it is better to ball-mill the tissues after gtrst blending them in an ultra-turrax for 5 min. We found that unless the tissues were ball-milled, it was very difficult to remove the starch completely from starch-rich tissues (e.g. potatoes and cereals). The relative merits of the procedure are discussed in Phytochemistry 1975 14: 1011.1017and 1978, 17: 745-752.

Dr. Walther (Grfinbach/FRG):

Is there any need for the bile salts to be adsorbed to the dietary fibre components? Further, you studies may have a bearing on serum cholesterol levels, but have you considered the possibility of degradative effects in the digestive tract?

Dr. Selvendran:

Dietary fibre binds bile salts by virtue of certain polymers which it contains. Physiolo- gical studies in man have shown that cereal fibre and vegetable (or fruit) fibre have different properties. Cereal fibre increases faecal weight and so far has been shown to have only a slight effect on serum cholesterol levels, but vegetable fibre has hypocho- lesterolemic properties. The results of our investigations with purified fibre preparations from vegetables shows which groups of polysaccharides in the fibre are responsible for binding bile salts and throws some light on the mechanism of binding. The enhanced elimination of the bile salts by dietary fibre rich in pectins could be a means of removing hepatic cholesterol resulting in lower blood cholesterol levels.

Dietary fibre constituents as well as bile salts are degraded by micro-organisms in the human digestive tract, especially in the large intestine. The work by Cummings et al. (1978) shows that dietary fibre is largely metabolised in the human gut and dietary

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pectin completely so. This is presumably why pectic substances have little effect on faecal weight. However, the bulk of the 'free' bile salts are reabsorbed in the ileum and a large proportion of those which survive into the large intestine (bound to the fibre) are likely to be excreted.

Dr. Gormley {Dublin/Ireland):

Have you done any tests on the strength of binding of bile salts by cell wall material of parenchymatous, lignified and cutinised tissues?

Dr. Selvendran:

Yes, we have done some experiments which throws light on the strength of binding of bile salts by vegetable fibre preparations. For example, 30-40% of the Na cholate bound by fibre at pH4 could be desorbed by increasing the pH to 8. Also, absolute alcohol and 8M urea desorb about 90% and 40%respectively of the Na cholate bound at pH 4.

Mrs. It. M. Stasse-Wolthuis [Wageningen/The Netherlands)

Pectic substances may lower the level of serum cholesterol by binding bile salts. How- ever, the results of our experiments, on the effects of 'natural' high-fibre diets, suggest that the effects on serum cholesterol cannot be explained completely by an increase in excretion of faecal steroids. Possibly, when steroid excretion increases, the synthesis of cholesterol in liver increases as well.

Dr. Selvendran:

What you say may well be true and the hypothesis warrants further work. All that I would like to say is that our work throws some additional light on the hypocholestero- lemic effects of vegetable fibre.

Author's address: Dr. R.R. Selvendran ARC Food Research Institute Colney Lane Norwich, NR4 7UA GB

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the binding of bile salts by vegetable fibre

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