Accepted Manuscript
Molecular interactions of a model bile salt and porcine bile with (1,3;1,4)-β- glucans and arabinoxylans probed by13C NMR and SAXS
Purnima Gunness, Bernadine M. Flanagan, Jitendra P. Mata, Elliot P. Gilbert, Michael J. Gidley
PII: S0308-8146(15)30104-7 DOI: http://dx.doi.org/10.1016/j.foodchem.2015.10.104 Reference: FOCH 18299
To appear in: Food Chemistry
Received Date: 26 May 2015 Revised Date: 23 September 2015 Accepted Date: 21 October 2015
Please cite this article as: Gunness, P., Flanagan, B.M., Mata, J.P., Gilbert, E.P., Gidley, M.J., Molecular interactions of a model bile salt and porcine bile with (1,3;1,4)-β-glucans and arabinoxylans probed by13C NMR and SAXS, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.10.104
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Title: Molecular interactions of a model bile salt and porcine bile with (1,3;1,4)-β-
2 glucans and arabinoxylans probed by13C NMR and SAXS
3 Authors: Purnima Gunnessa, Bernadine M. Flanagana, Jitendra P. Matab, Elliot P.
4 Gilbertb, and Michael J. Gidleya*
5 Address of laboratories: aARC Centre of Excellence in Plant Cell Walls and
6 Centre for Nutrition and Food Sciences
7 Queensland Alliance for Agriculture and Food Innovation,
8 The University of Queensland, Hartley Teakle Building
9 St Lucia, Queensland, 4072, Australia
10 bBragg Institute, Australian Nuclear Science and Technology
11 Organisation (ANSTO), Locked Bag 2001, Kirrawee DC, NSW,
12 2232, Australia
13 Email addresses of authors: Purnima Gunness: [email protected]
14 Bernadine M. Flanagan: [email protected]
15 Jitendra P. Mata: [email protected]
16 Elliot P. Gilbert: [email protected]
17
18 *Corresponding author: Prof. Michael Gidley
19 Centre for Nutrition and Food Sciences,
20 Queensland Alliance for Agriculture and Food Innovation,
21 The University of Queensland, Hartley Teakle Building
22 St Lucia, Queensland, 4072, Australia
24 Telephone: +61 7 3365 2145
25 Fax: +61 7 3365 1188 26 Abbreviations:
27 - AX Arabinoxylan
28 - βG (1,3:1,4)-beta-D-glucan
29 - BS Bile salts
30 - GC Glycocholate
31 - GDC Glycodeoxycholate
32 - GCDC Glycochenodeoxycholate
33 - GHC Glycohyocholate
34 - GHDC Glycohyodeoxycholate
35 - LMW Low molecular weight
36 - MMW Medium molecular weight
37 - HMW High molecular weight
38 - PB Porcine bile
39 - TC Taurocholate
40 - TDC Taurodeoxycholate
41 - TCDC Taurochenodeoxycholate
42 - THC Taurohyocholate
43 - THDC Taurohyodeoxycholate
44 - TSP 3-(trimethyl silyl) propionic acid-d4
45 - NMR Nuclear Magnetic Resonance
46 - SAXS Small angle X-ray scattering
47 Key words: (1,3:1,4)-β-D-glucan, arabinoxylan, bile salt, 13C NMR, SAXS
48 Chemical compounds: Glycochenodeoxycholic acid sodium salt (PubChem CID:
49 23696298); Taurochenodeoxycholic acid sodium salt (PubChem CID: 23687512)
50 51 Abstract
52 Two main classes of interaction between soluble dietary fibres (SDFs), such as (1,3:1,4)- β-
53 D- glucan (βG) and arabinoxylan (AX) and bile salt (BS) or diluted porcine bile, were
54 identified by 13C NMR and small angle X-ray scattering (SAXS). Small chemical shift
55 differences of BS NMR resonances were consistent with effective local concentration or
56 dilution of BS micelles mostly by βG, suggesting dynamic interactions; whilst the reduced 57 line widths/intensities observed were mostly caused by wheat AX and the highest molecular
58 size and concentrations of βG. SAXS showed evidence of changes in βG but not AX in the
59 presence of BS micelles, at >13 nm length scale consistent with molecular level interactions.
60 Thus intermolecular interactions between SDF and BS depend on both SDF source and its
61 molecular weight and may occur alone or in combination. 62 1. Introduction
63 It is accepted that viscous cereal soluble dietary fibres (SDFs), such as (1,3:1,4)- β-D-glucan
64 (βG) found in oats (Ripsin, Keenan, Jacobs, Elmer, Welch, Vanhorn, et al., 1992) and barley
65 (Newman, Newman, & Graham, 1989) and arabinoxylan (AX) present in wheat and rye, may
66 help lower serum cholesterol. One of the possible mechanisms (Gunness & Gidley, 2010) is
67 molecular interactions between SDF and bile salts (BS) causing an excess elimination of BS
68 in the faeces, resulting in the recruitment of cholesterol to synthesise replacement BS, thereby
69 lowering serum cholesterol levels (Ellegard & Andersson, 2007). In vitro study of the
70 interactions between SDF and BS that may occur in the small intestine is important for
71 understanding how SDF causes a decrease in serum cholesterol as reported in clinical
72 (Fernandez, 2001; Queenan et al., 2007) and animal (Anderson, Deakins, Floore, Smith, &
73 Whitis, 1990; Turley, Daggy, & Dietschy, 1991) investigations.
74 BS are acid sterols synthesised solely from cholesterol in the liver and stored in the gall
75 bladder where they constitute a major component of bile. These molecules readily aggregate
76 above a critical micellar concentration to form micelles. Their primary function in the
77 digestive tract is to solubilise and transport hydrophobic entities, such as lipophilic nutrients.
78 In humans, the dominant BS are the tauro- and glyco- conjugates of cholate, deoxycholate
79 and chenodeoxycholate; while in pigs the most abundant species are hyocholate,
80 hyodeoxycholate, chenodeoxycholate and small amounts of the tauro- and glyco- conjugates
81 of deoxycholate (Kandrac et al., 2006; Kuhajda, Kandrac, Trajkovic, & Hranisavljevic, 1998)
82 (Fig. 1.).
83 Insert Figure 1 here
84 Cereal SDFs, such as βG and AX, are polysaccharides of high molecular weight and are
85 capable of forming gels under certain conditions (Lazaridou & Biliaderis, 2007). βG
86 molecules are linear unbranched homopolymers generally formed by blocks of 2 or 3 β- 87 glucosyl residues joined by (1,4)-β linkages and separated by single (1,3)-β linkages (Buliga,
88 Brant, & Fincher, 1986). The presence of the (1,3)-β linkages renders the βG molecule
89 flexible, stable and water soluble (Bohm & Kulicke, 1999). The presence of a mixture of
90 linkage types with a probable random distribution (Buliga et al., 1986) of cellotriose and
91 cellotetraose units linked by (1,3)-β linkages limits intermolecular helix formation giving rise
92 to predominantly random coil conformations (Gidley & Nishinari, 2009). AX is a
93 heteropolymer consisting of a xylopyranose (Xyl) backbone linked by β-(1-4) linkages to
94 which substituents of α-L-arabinofuranose (Ara) are attached by (1-3) and/or (1-2) linkages.
95 In cereals, the pattern of Ara substitution, the ratio of Ara/Xyl and the molecular size all vary.
96 This confers differences in physicochemical properties, such as viscosity, gel-formation and
97 intermolecular associations between cereal AXs (Izydorczyk & Biliaderis, 1995; Izydorczyk
98 & Dexter, 2008).
99 Three hypotheses have been postulated to explain how SDFs interact with BS in the small
100 intestine causing an excess loss of BS (Gunness & Gidley, 2010). In one hypothesis, SDF
101 intervenes at a macro-scale increasing the absorptive resistance of the unstirred water layer
102 lining the intestinal epithelium. The other two hypotheses invoke interactions of SDF with BS
103 at a molecular level by direct interaction and/or at a meso-scale by intermolecular
104 associations of polymer strands which reduces the mobility of the BS micelles. In preliminary
105 work, we studied the last two hypotheses using a single BS molecule,
106 taurochenodeoxycholate (TCDC), to model the interactions of whole bile with single samples
107 of barley βG and wheat AX (Gunness, Flanagan, & Gidley, 2010). From this 13C NMR study,
108 we deduced that SDFs can interact with BS micelles either by forming dynamic complexes
109 with the micelles (as indicated by systematic chemical shift changes in BS resonances) or by
110 trapping BS micelles in a local aggregate structure resulting in reduced BS mobility and
111 consequent broader, less intense BS resonances (Gunness et al., 2010). 112 We now report a comprehensive study on the interactions between SDFs and BS using both
113 13C NMR (molecular level) and small angle X-ray scattering (SAXS) (> 13 nm (Q~0.05 Å-1)).
114 Three different molecular weights (MWs) and two different sources for each of βG (barley
115 and oat) and AX (wheat and rye) were used to identify interactions with a model BS,
116 glycochenodeoxycholate (GCDC) using 13C NMR spectroscopy. These studies were then
117 extended for a barley βG and a wheat AX with (a) small angle X-ray scattering (SAXS) to
118 probe the micellar and polymer structure and interactions in the presence of GCDC, and (b)
119 NMR to probe interactions with whole porcine bile. The results show that there were two
120 main types of intermolecular interaction, dynamic molecular interaction and entrapment of BS
121 micelles by the SDF as well as a combination of both depending on the SDF source and its
122 molecular weight and concentration.
123 2. Materials and methods
124 2.1. Materials 125 126 The sodium salt of GCDC, 3-(trimethyl silyl) propionic acid-d4 (TSP) and D2O (99 atom
127 %D) were purchased from Sigma Aldrich Pty. Ltd. (Castle Hill, NSW, Australia). The
128 sodium salt of TCDC was purchased from Calbiochem, Merck Pty. Ltd. (Victoria, Australia).
129 Barley βG of low molecular weight (LMW) with manufacturer’s kinematic viscosity
130 specifications at 1% w/v of 10 cSt (Lot No. 10401); medium molecular weight (MMW), 28
131 cSt (Lot No. 10301) and high molecular weight (HMW), 100 cSt (Lot No. 90501); and wheat
132 AX of LMW, 2 cSt (Lot No. 90201); MMW, 29 cSt (Lot No. 40301) and HMW, 47 cSt (Lot
133 No. 80601), oat βG, 69 cSt (Lot No. 80608) and rye AX, 33 cSt (Lot No. 20601a) were
134 purchased from Megazyme International (Bray, Ireland). Molecular weight data for these
135 samples have been reported elsewhere (Pitkanen, Virkki, Tenkanen, & Tuomainen, 2009;
136 Shelat, Vilaplana, Nicholson, Gidley, & Gilbert, 2011) and are summarised in Supplementary
137 Material (Supp.) Table 1. Phosphate buffer (0.133 M, pH 6.2) was made from 0.133 M 138 Na2HPO4 and 0.133 M KH2PO4 both of analytical grade in deionised water. Bile was
139 collected from the gall bladder of anesthetised healthy large white pigs (animals were fed
140 control diets as described in Belobrajdic et al. (Belobrajdic et al., 2012)) and stored at -80ºC
141 for further use.
142 2.2. Sample preparation
143 2.2.1. (1,3:1,4)-β-D-glucan and arabinoxylan solution preparation
144 βG and AX solutions at 0.5% w/v and 2% w/v were made by adding weighed amounts of the
145 polymers to 10 ml of boiling 0.133 M phosphate buffer (pH 6.2) in sealed vials and
146 maintained under constant agitation with a magnetic stirrer overnight at 80-90°C. Freshly
147 made and solutions stored at 4°C for up to 4 weeks were used to check if storage had any
148 influence on the structure of the polymers. As the 13C NMR spectra showed no difference, the
149 solutions kept at 4°C were used for the different experiments to minimise variations between
150 samples.
151 2.2.2. NMR sample preparation
152 Two hundred microlitres of 20 mM GCDC in the phosphate buffer was added to 1 ml of the
153 polymer solutions of 0.5% w/v and 2% w/v respectively. The final concentration of the
154 polymer was thus 0.42% w/v and 1.67% w/v respectively. Pure porcine bile was diluted 5
155 times to approximate the NMR signal intensity of 20 mM GCDC (ca 0.9% w/v). To assist in
156 NMR assignment, 10 mM of TCDC was added to a 1 in 10 dilution of pure bile. The
157 mixtures were vortex mixed at high speed and incubated for 2 h in a water bath maintained at
158 37°C under constant agitation (84 rpm) (Dongowski, 2007) to allow for structural
159 equilibration. TSP in D2O (100 µl, 1.2 mg/ml) was added to 600 µl of the mixture. Each
160 sample was freshly prepared prior to the NMR experiment. Concentrations of SDF and
161 BS/bile were chosen to cover the expected range for diets rich in SDF (Hofmann, 1989;
162 Laerke, Pedersen, Mortensen, Theil, Larsen, & Knudsen, 2008; Smith & Tucker, 2011) and 163 BS concentrations in the small intestine. GCDC concentrations in all experiments were above
164 the critical micelle concentration.
165 2.2.3. SAXS sample preparation
166 To investigate the potential interactions between BS and SDF and the conformation of their
167 mixed aggregates, three concentrations of SDFs were studied by SAXS in the presence and
168 absence of 3.33, 16.7 and 100 mM GCDC. 1% w/v and 2% w/v βG and AX stock solutions
169 were made as described for NMR samples. These solutions were then mixed either with
170 0.133 M phosphate buffer (pH 6.2) and/or with 200 mM GCDC (ca 9% w/v) to produce 17
171 different solutions as shown in Supp. Table 2. The mixtures were vortex mixed at high speed
172 and incubated for 2 h at 37°C with regular agitation (Dongowski, 2007).
173 2.3. 13C NMR experiments
174 13C NMR titration is a well-established method for studying molecular-level interactions,
175 such as those between SDFs and bile salts in aqueous solution (Gunness et al, 2009;
176 Mikkelsen, Cornali, Jensen, Nilsson, Beeren, & Meier, 2014). To further check for
177 consistency of results, samples were run consecutively with the NMR probe heated at 37°C
178 on a 500-MHz spectrometer and at 25°C on a 900-MHz spectrometer. As no difference was
179 detected in the spectra, all 13C NMR experiments were performed on a Bruker Biospin
180 Avance 900-MHz NMR spectrometer at 25°C for faster acquisition. The spectrometer was
181 operated at 225 MHz for 13C, with a 5 mm TCI cryoprobe equipped with a shielded z-
182 gradient and controlled by XWIN-NMR software (Zürich, Switzerland). Each 13C NMR
183 spectrum was obtained with a total acquisition time of 4 h under the following conditions:
184 9000 scans, 90° pulse, an acquisition time of 0.66 s, and a relaxation delay of 1 s. The spectral
185 width was 50 kHz, time domain 64 K and a line broadening of 5 Hz was applied. TSP was
186 used as an internal chemical shift standard (0 ppm) and D2O acted as a lock for the 187 spectrometer. Spectra were processed with MestReNova software version 5.20 (2007,
188 Mestrelab Research S.L, Spain).
189 2.4. SAXS experiments
190 Small angle scattering studies allow investigation of interactions between components such
191 as SDFs and bile salts in aqueous solutions at length scales of 10 nm upwards (Lopez-Rubio
192 & Gilbert, 2009; Lopez, Samseth, Mortensen, Rosenqvist, & Rouch, 1996). All solutions
193 (Supp. Table 2) were loaded into 2 mm sealed quartz capillaries and the scattering was
194 measured for 60 minutes at room temperature. For absolute scale calculation, the scattering
195 from empty capillaries and water-filled capillaries were also collected. SAXS measurements
196 were obtained using a Bruker NanoStar SAXS instrument equipped with Vantec 2000 area
197 detector (effective pixel size 54 µm) and pin-hole collimation for point focus geometry with a
198 wavelength of 1.54 Å. The optics and sample chamber were under vacuum to minimize air
199 scattering. The sample to detector distance was chosen to be 700 mm, which provided a Q-
200 range from 0.014 to 0.430 Å-1, where the magnitude of the scattering vector, Q, is defined as:
4π Q = sinθ 201 λ
202 and where λ is the wavelength and θ is the scattering angle. SAXS curves were normalized to
203 sample transmission, radially averaged and background-subtracted using Bruker AXS
204 software 4.1.30 and Igor (Wavemetrics, Lake Oswego, Oregon, USA). Scattering was placed
205 on an absolute scale with respect to water as reported by Orthaber et al. (Orthaber,
206 Bergmann, & Glatter, 2000). Scattering curves are plotted as functions of absolute intensity,
207 I, versus Q.
208 To assess the extent to which the observed scattering differed from that predicted from a
209 homogeneous mixtures of all components (i.e. in the absence of intra- or inter-particle 210 changes), ITotal is also plotted where this is defined as the sum of two terms, ISDF (scattering
211 from SDF) and IBS (scattering from BS):
212 ITotal = I SDF + I BS .
213 Any deviation of I from ITotal indicates conformational or intra-particle (form factor) or
214 interaction (structure factor) changes.
215 3. Results and discussion
216 3.1. 13C NMR
217 Table 1 summarises the changes observed in the GCDC signals in the presence of different
218 sources and viscosity grades of βG and AX. The SDFs interacted with GCDC in different
219 ways with two major behaviours: most βGs (except MMW barley βG) caused changes in
220 chemical shifts, while decreases in GCDC resonance intensities relative to TSP were
221 observed in the presence of all AXs. However, for HMW barley and oat βG, LMW wheat AX
222 and rye AX, both chemical shift changes and decreases in GCDC resonance intensities
223 relative to TSP were observed. Very small relative chemical shifts (<0.02 ppm) were also
224 observed in the presence of MMW wheat AX and MMW barley βG, but these were smaller
225 than for other AX and βG samples, and have therefore not been classified as chemical shift
226 changes. No systematic change in chemical shifts and/or decreases in intensities were seen in
227 the βG and AX signals for any sample in the presence or absence of bile salts.
228 Insert Table 1 here
229 Full details of chemical shift changes, together with peak height and area data are given in the
230 Supp. Tables 3-10. Chemical shift changes were found to be of two general types (Table 1).
231 One involved typically upfield shifts (i.e. to lower ppm values) for resonances with high ppm
232 values (> 50 ppm) and typically downfield shifts for resonances at < 50 ppm. The other was 233 the opposite with the transition between upfield and downfield shifts again occurring at
234 around 50 ppm (Supp. Tables 3 and 4).
235 To illustrate the observed changes, Fig. 2A shows regions of the 13C NMR spectra of 20 mM
236 GCDC alone (1) and in the presence of 0.42% w/v (2) and 1.67% w/v (3) LMW barley βG
237 showing the assigned peaks for the GCDC molecule (Ijare, Somashekar, Jadegoud, & Nagana
238 Gowda, 2005). Numerous small changes in GCDC resonance chemical shifts are observed in
239 the presence of LMW barley βG as indicated by the dotted lines, without any apparent change
240 in relative intensity or line width compared with the TSP internal standard. Although all the
241 changes in Fig. 2A are downfield, other resonance shift changes are upfield, particularly
242 those at higher ppm values (Supp. Table 3). Interestingly, the relative chemical shift changes
243 in the presence of 0.42% w/v βG were greater than with 1.67% w/v βG.
244 Insert Fig. 2. here
245 In contrast, for HMW barley, GCDC chemical shift changes increased with increasing SDF
246 concentrations (Supp. Table 3), as did GCDC in the presence of oat βG (Fig. 2B). This is
247 consistent with our previous study on a different BS (TCDC) in the presence of MMW barley
248 βG which also showed that the relative chemical shift differences were greater with
249 increasing concentrations of βG (Gunness et al., 2010). Fig. 2B shows an example of the
250 results obtained with oat βG at 0.42% w/v (2) and 1.67% w/v (3) in the presence of GCDC.
251 The differences in chemical shifts increased and at the same time the intensities of the peaks
252 decreased for the higher βG concentration. In addition the peak intensities of GCDC signals
253 decreased with increasing concentrations for HMW barley βG, however the areas under the
254 peak decreased more in the presence of the oat βG with a greater effect for the 1.67% w/v
255 sample (Supp. Tables 6 & 8). In contrast to the interaction of MMW barley βG with TCDC
256 (Gunness et al, 2010), which showed similar chemical shift changes to those seen here for
257 GCDC with HMW βG, the chemical shift changes for GCDC in the presence of MMW 258 barley βG were very small (Tables 1 and Supp. 3). This illustrates the subtle nature of SDF-
259 BS interactions and the fact that details may vary with different BS’s.
260 In the presence of LMW wheat and rye AX (and to a lesser extent HMW wheat AX), both
261 changes in chemical shifts and decreases in intensities of GCDC resonances were observed
262 (Fig. 3), whereas for MMW wheat AX only decreases in signal peak heights and areas under
263 the peaks occurred (Supp. Fig. 1). The decrease in signal peak heights was greatest with
264 1.67% HMW AX while the greatest decrease in area under the signals was obtained with
265 1.67% LMW AX (Supp. Tables 5 & 7). In addition, LMW AX (and to a lesser extent HMW
266 AX) caused changes in chemical shifts in the GCDC signals which were mostly in the
267 opposite direction compared to rye AX and all βG samples (Supp. Tables 4 & 5).
268 Insert Fig. 3. here
269 In all cases, there were no systematic changes in polysaccharide resonance chemical shifts,
270 suggesting that the observed GCDC effects may be due to local structure variation within the
271 BS micelles. To test this, a series of GCDC concentrations was studied. Supp. Fig. 2 and
272 Supp. Table 9 show that there were systematic changes in chemical shifts, downfield for low
273 ppm (< 50 ppm) and upfield for high ppm (> 50 ppm), with increasing concentrations that
274 were in the same direction as those seen for the addition of LMW, and HMW barley βG, as
275 well as rye AX. This suggests that the addition of these SDFs increases the effective local
276 concentration of BS micelles. Conversely, the addition of increasing amounts of oat βG,
277 LMW, and HMW wheat AX change the chemical shifts upfield for low ppm (< 50 ppm) and
278 downfield for high ppm by reducing the effective local concentration of BS micelles,
279 presumably by mixing sufficiently thoroughly to cause separation of adjacent micelles.
280 To test whether similar interactions occur in whole bile, NMR spectra of diluted porcine bile
281 were obtained in the presence of MMW barley βG and MMW wheat AX (Fig. 4). Some
282 similarities, but also some differences, in behaviour are seen for the bile salts in diluted 283 porcine bile compared with the pure BS (TCDC (Gunness et al., 2010) and GCDC) studied in
284 the presence of MMW barley βG and MMW wheat AX. The NMR spectrum of porcine bile
285 contains resonances that can be assigned to the expected range of related sterol skeletons and
286 tauro/glyco-conjugates (Fig.1). Diluted pure bile was spiked with TCDC to facilitate
287 resonance assignment as most of the BS signals in whole bile overlap. All major peaks were
288 assigned to GCDC, TCDC, GC, TC, GDC, TDC, HDC and HC according to literature values
289 (Gowda et al., 2006; Iida et al., 1989; Ijare et al., 2005; Vandelli et al., 2000) and GCDC
290 NMR spectra (Supp. Table 4). Resonances for equivalent carbons in glyco- and tauro- BS
291 have very similar chemical shifts apart from C-25 and 26 which represent the tauro/glyco-
292 region of the molecule (Fig.1).
293 Insert Fig. 4. here
294 Table 10 (Supp.) summarises the effects of 1.67% w/v MMW barley βG and MMW wheat
295 AX on diluted porcine bile respectively. βG caused smaller chemical shift changes in the BS
296 resonances than for the pure GCDC or TCDC, but had a similar non-effect on peak heights
297 and intensities as the pure BS. AX caused similar decreases in peak heights and/or area under
298 the peaks in both GCDC or TCDC and diluted porcine bile. Some small relative chemical
299 shifts (similar to those of βG) were also found in the presence of AX (Supp. Table 10). Fig. 4
300 represents different regions of the spectra obtained for TCDC-spiked porcine bile alone and
301 in the presence of 1.67% w/v MMW βG (Figs 4A & 4B) and AX (Figs. 4C & 4D). In the
302 presence of AX, some peaks, for example C-21 GC/TC/GDC/TDC (Fig. 4C), became broader
303 and merged with C-21 TCDC/GCDC while others, such as C-15 TCDC (Fig. 4D), split into 2
304 peaks. This resulted in a decrease in measured apparent signal intensities. Comparison of the
305 same region with βG samples (Figs. 4A & 4C), showed that the peaks of the GCDC signals
306 retained their shape and only (small) chemical shift changes were observed. Some C-atoms
307 (e.g. C-9 and 14 in TDC/GDC, TCDC/GCDC in porcine bile) shifted in the same directions 308 as pure GCDC in the presence of 1.67% w/v βG (Supp. Table 3). The values of the area
309 under the peaks were variable for AX, probably due to variable line broadening as well as
310 overlapping of signals.
311 3.2. SAXS
312 To further probe the interactions between SDFs and BS, the scattering from GCDC, MMW
313 barley βG or MMW wheat AX, and their mixtures were measured. Fig. 5 (upper panel)
314 shows the SAXS from βG (0.1 and 0.5 % w/v) in the presence and absence of 100 mM (ca
315 4.5% w/v) GCDC. The scattering from GCDC is also shown, and represents a typical
316 scattering profile from BS micelles (Lopez-Rubio & Gilbert, 2009; Lopez, Samseth,
317 Mortensen, Rosenqvist, & Rouch, 1996). Itotal, which corresponds to the numerical sum of the
318 scattering from both solutions, has also been plotted, and represents the total theoretical
319 intensity of βG and GCDC when they are present in solution in the absence of structural
320 changes and/or interactions. At 0.5% w/v, βG in the presence of 100 mM GCDC showed a
321 reduced (~40%) intensity at low-Q compared to Itotal. However, there was no change beyond
322 Q > ~0.03 Å-1 (<13 nm), suggesting no variation in the structure of the GCDC micelles or in
323 the shorter range structure of the βG which was probed in NMR experiments. This behaviour
324 could arise as a result of changes at >13 nm due to conformational changes in the βG, inter-
325 chain interactions, or βG-GCDC micelle interactions.
326 Insert Fig. 5. here
327 We have also found that scattering at 0.1% w/v βG in the presence of 100 mM GCDC
328 exhibited little difference to Itotal. This could indicate that, at this much lower concentration,
329 there is insufficient sensitivity to discern the βG-GCDC interaction although we cannot
330 exclude the possibility of a concentration-dependent interaction or scattering differences
331 outside the accessible Q range. These results are in line with the 13C NMR results; indeed, in 332 the presence of a low concentration (0.08% w/v) barley βG, no chemical shifts changes were
333 observed in the TCDC resonances (Gunness et al., 2010). In contrast, for the AX system we
334 found that in the presence of GCDC the scattering of the mixture was indistinguishable to
335 that of ITotal (Fig. 5 lower panel), suggesting no conformational changes of AX, nor
336 interchain, nor SDF-BS interactions. This is consistent with observations from the 13C NMR
337 results (Table 1 & Supp. Table 4) where MMW wheat AX did not cause chemical shift
338 changes. The origin of the scattering differences observed (or lack thereof) does not appear to
339 be associated with changes in the BS micelle dimensions in the presence of SDF evident from
340 the lack of scattering change at higher Q (thus <13 nm structure). However, scattering
341 differences may be related to either a change in polymer conformation in the presence of BS
342 micelles, inter-polymer or polymer-micelle interactions, thus detailed modelling is
343 problematic. To decouple these effects, Small-Angle Neutron Scattering (SANS) experiments
344 may be valuable using solvent contrast variation in which the micelle is contrast matched
345 (Lopez-Rubio & Gilbert, 2009).
346 To further assess concentration effects, scattering from a more concentrated SDF system,
347 namely 1.7% w/v MMW wheat AX and MMW barley βG, in the presence and absence of
348 much lower concentrations (either 3.33 or 16.7 mM) of GCDC was also recorded. The results
349 (Supp. Fig. 3) show a similar reduction in low q scattering at Q ~ 0.05 Å-1 (at >13 nm length
350 scale) for βG but not AX in the presence of BS as observed at 0.5% w/v (Fig. 5). The
351 different behaviour is therefore consistent across a broad concentration range of both SDF
352 and BS; the influence of BS concentration shows that there was a greater effect at 16.7 mM
353 than 3.33 mM on the low Q scattering observed with βG. The SAXS data, therefore, point to
354 changes on a length scale >13 nm for MMW barley βG + BS systems but not MMW wheat
355 AX + BS systems. If this effect arises from a conformational change of the polymer, it is
356 worthy of note that it is occurring on a length scale comparable to and greater than that of the 357 reported persistence length 3-5 nm of the two polymers (Gomez, Navarro, Garnier, Horta, &
358 Carbonell, 1997; Picout & Ross-Murphy, 2002) but not at the shorter length scale probed by
359 NMR.
360 3.3. SDF – BS interaction mechanisms
361 3.3.1. General 362 363 Blood cholesterol-reducing properties of SDF have been attributed to increased intestinal
364 luminal viscosity by HMW oat βG compared to hydrolysed βG in a human trial (Wolever,
365 Tosh, Gibbs, Brand-Miller, Duncan, Hart, et al., 2010). However, the nature of the
366 interactions between the viscous SDF polymers and BS in the intestinal lumen is difficult to
367 assess in vivo. Thus, in vitro experiments have been carried out by various groups to
368 investigate these interactions in simple systems to help understand the health conferring
369 benefits of SDFs. In an attempt to explain how cereal SDFs interact with BS, an in vitro
370 experiment showed that MMW barley βG and wheat AX reduce the diffusion rate of 20 mM
371 TCDC micelles and whole bile across a semi permeable membrane. These results suggest that
372 the polymers restrict the motion of micelles by direct interactions, viscosity effects, and/or by
373 forming aggregates around them (Gunness, Flanagan, Shelat, Gilbert, & Gidley, 2012).
374 However, the mechanisms by which SDF bind to BS remain unclear (Kahlon, Chiu, &
375 Chapman, 2009), despite the central role proposed for these in leading to cholesterol
376 reduction.
377 In this study, we used a simple mono-molecular BS micelle to model the interactions of the
378 numerous bile salts found in bile, as well as whole porcine bile. Our preliminary 13C NMR
379 experiments showed that the viscous nature of the SDF is not the only factor restricting the
380 mobility of the BS micelles and we concluded that MMW wheat AX and BS micelles can
381 interact at a meso-scale by forming a local network around the micelles and that MMW 382 barley βG can interact dynamically with BS micelles causing BS chemical shift changes
383 without any BS line width changes or apparent βG resonance changes (Gunness et al., 2010).
384 In this present work, extended to cereal SDF of different molecular weights and origins, the
385 13C NMR results showed that most of the βGs caused chemical shift changes in GCDC and
386 whole porcine bile, while all of the AX samples decreased the intensities of the BS
387 resonances but with no apparent change in the conformation of both polymer. However, at a
388 longer length-scale (>1 nm) the SAXS results showed that the apparent dimensions of only
389 the βG polymer but not AX was affected by the presence of the micelles. Moreover, 13C
390 NMR showed that with high MW SDFs as well as low MW wheat AX, a combined effect of
391 both mechanisms, dynamic molecular interactions and network formation was seen in the
392 GCDC system.
393 3.3.2. Molecular interactions
394 Chemical shift differences for the GCDC signals in the presence of many SDFs (Supp. Table
395 3) suggest the possibility that the polymer and the BS micelles might be bound or complexed
396 by H-bonds and/or van der Waals forces. However, as the shift changes were only small (<
397 0.11 ppm) and there were no systematic changes in SDF shifts or line widths, any interactions
398 are considered to be dynamic and not permanent. The SAXS data provide evidence of non-
399 additive behaviour for MMW barley βG and GCDC at >13 nm (Q ~ 0.05 Å-1), affecting the
400 apparent dimensions of the polymer without affecting BS micelle size. Together with the
401 NMR evidence this is consistent with a close association of the two components on average
402 with a rapid equilibrium between free and bound BS. The lack of any marked chemical shift
403 changes for βG (or AX) resonances in the presence of BS or porcine bile suggests that any
404 changes in the average local chain conformation ( 405 SAXS data suggests a change in overall βG dimensions at >13 nm length scale (Q ~ 0.05 Å-1) 406 by closer association between the βG coiled structures. Therefore, the two techniques in 407 combination provide evidence that both BS and βG are affected by each other in solution at 408 different length scales of measurement. 409 The chemical shift changes in the model BS in the presence of βG occur for sites across the 410 molecule (Fig. 1) suggesting that the whole micelle is affected either by direct interaction 411 with carbohydrate chains or by a change in internal micelle organisation. The smaller 412 chemical shift changes seen in the presence of whole porcine bile, suggests that the more 413 complex micelles in bile are not affected by the βG to the same extent. The pattern of BS 414 chemical shift changes in the presence of most SDFs is similar to that observed for increasing 415 concentrations of BS alone (Supp. Table 9), consistent with SDFs causing an apparent 416 increase in effective local BS concentration, probably through excluded volume effects. 417 However, the lack of change in SAXS from the micelles shows that any concentration effect 418 does not markedly change the overall micelle dimensions at <13 nm length scale. 419 There are three other interesting findings connected with chemical shift changes. One is the 420 greater shift changes for 0.42% vs 1.67% LMW barley βG (Fig. 2A), which suggests that 421 there is a greater effect on micelles at the lower concentration. This might be due to a low 422 saturation concentration of LMW βG for BS interaction, such that the effect is diluted out at 423 1.67% w/v βG. A second interesting finding is the chemical shift changes seen for LMW and 424 HMW wheat AX as well as oat βG, which are generally in the opposite direction at 425 individual carbon sites compared to other SDFs (Supp. Tables 3 & 4). This suggests that 426 these SDFs cause an effective dilution of BS micelles. We suggest that this is caused by 427 intimate mixing (without specific association) such that the SDF effectively screens micelles 428 from each other. The difference in behaviour between oat and barley βG could be due to their 429 fine structure. Indeed, their DP3:DP4 ratio is approximately 2 and 3 respectively (Lazaridou, 430 Biliaderis, Micha-Screttas, & Steele, 2004). This means that oat βG has more of the longer 431 cellulose-like DP4 segments resulting in closer intermolecular associations between the oat 432 βG chains allowing better mixing with the micelles. A third finding is that rye AX shows a 433 different pattern of chemical shift changes to the three examples of wheat AX. This is 434 interesting as the arabinose to xylose ratio of rye AX (1:2.2 from 1H NMR) is similar to that 435 of wheat AX (1:2.1 from 1H NMR). For rye AX, it is possible that the different detailed 436 molecular structure may play a role. 1H NMR data shows that wheat AX contains 62% 437 unsubstituted xylose, 19% mono-substituted (mostly at O-3) and 19% di-substituted (at both 438 O-2 and O-3), most probably non-randomly distributed (Izydorczyk & Biliaderis, 1994). We 439 propose that the bulky di-substitution effectively shields the xylan backbone from interacting 440 with the BS micelles. In contrast, rye AX has a much lower level of di-substitution (8%) 441 which may allow a closer approach of micelles to the xylan backbone over sufficiently long 442 stretches to stabilise interaction. A typical micelle diameter of ~5 nm corresponds to 443 approximately 10 backbone xylose residues in AX. 444 3.3.3. Micelle trapping 445 A characteristic of all wheat AX systems was the ability to broaden and reduce the intensity 446 of BS resonances in model micelles as well as intact porcine bile. The fact that line width 447 effects (Supp. Table 6) did not necessarily correlate with integrated signal intensity (Supp. 448 Table 8) suggests that some BS micelles are sufficiently immobilised that individual 449 resonance line widths are too broad to be observed, whereas some micelles have intermediate 450 mobility resulting in broadened but still detectable signals. This line broadening is not a 451 general effect of SDF solution viscosity as no detectable line broadening was found for e.g. 452 MMW βG which has a greater viscosity than either LMW or MMW wheat AX. It is possible 453 that some infiltration by AX side chain arabinose residues into the hydrophilic head-group 454 regions of BS micelles may occur, explaining why AX samples are more prone to result in 455 BS resonance line broadening than non-branched βG samples. 456 It has recently been shown that solutions of both wheat and rye AX show variable amounts of 457 local aggregation that is sufficient to lead to unusual diffusion properties for a 70 kDa dextran 458 probe with a hydrodynamic radius of ca 6 nm (similar in dimensions to BS micelles) (Shelat 459 et al., 2010). It was proposed that local AX aggregation effectively prevents the dextran from 460 accessing regions enclosed by aggregates leading to faster diffusion through the remaining 461 accessible solution. We propose that BS micelles can access regions enclosed by aggregates 462 but are effectively immobilised for long enough to result in NMR line broadening. AX 463 aggregation does not seem to be affected by bile or model BS as there is no effective change 464 in either polymer dimensions from SAXS nor NMR line widths for AX in the presence of BS 465 micelles. 466 3.3.4. Molecular interaction and micelle trapping 467 In the presence of LMW and HMW wheat AX and oat βG, both changes in chemical shifts 468 and decreases in GCDC resonance intensities were observed (Table 1). It is striking that these 469 three samples are the only ones for which the chemical shift changes (upfield for < 50 ppm 470 and downfield for > 50 ppm) were equivalent to a dilution of bile salt micelles. This suggests 471 that intimate mixing and dynamic interaction of these SDFs with BS micelles, sufficient to 472 separate micelles from each other, is also associated with a decrease in mobility of micelles. 473 4. Concluding remarks 474 Through studying the interactions of a wide range of βG and AX samples with a model bile 475 micelle system by 13C NMR, both dynamic interaction leading to effective local increases or 476 decreases in BS concentration (characteristic of, but not restricted to, βG) and trapping of 477 micelles within polysaccharide aggregates (characteristic of, but not restricted to, AX) are 478 inferred. SAXS data are consistent with these processes with BS interaction shown to directly 479 affect βG dimensions at >13 nm length scale (Q ~ 0.05 Å-1), whereas BS micelles did not 480 change the SAXS behaviour observed for AX, showing that the trapping effect occurs 481 quickly (SAXS providing a time-averaged measurement of structure) or operates over a 482 longer length scale outside the accessible SAXS instrument range. Different AX and βG, and 483 whole porcine bile compared with model BS, show various combinations of the two 484 characteristic interaction types, with observations explicable in terms of variations in 485 molecular structure or size. This diversity of behaviour is of particular relevance to 486 cholesterol-reducing ability as it is high molecular weight (oat in an intervention trial 487 (Wolever et al., 2010) and barley in an animal experiment (Bengtsson, Aman, Graham, 488 Newman, & Newman, 1990) βG that is most associated with beneficial plasma cholesterol 489 effects. In this study, both oat and HMW barley βG showed NMR evidence for dynamic 490 interaction (Supp. Table 3) but the chemical shift changes were in opposite directions and oat 491 βG resulted in BS resonance line broadening, whereas HMW barley βG did not. In this 492 context it is interesting that the rye AX behaved similarly to HMW and LMW barley βG; and 493 HMW wheat AX behaved similarly to oat βG. Overall, although systematic effects were 494 obtained, all potential interactions appear to be weak and readily reversible, and no 495 interactions with sufficient permanence or strength to realistically survive passage through 496 the small intestine were found. This study therefore did not find any convincing evidence to 497 support the current lead hypothesis for cholesterol-lowering effects in blood via enhanced 498 excretion of bile salts by binding to SDF in the small intestine. 499 Acknowledgements 500 Funding was made available from the Australian Flagship Collaborative Research Program, 501 provided to the High Fibre Grains Cluster via the Food Futures Flagship. The authors would 502 like to thank Dr Gregory Pierens for assistance with the 900-MHz NMR spectrometer. 503 Supporting Information Available 13 504 Tables of chemical shifts, relative heights and integrated areas of C NMR resonances for 505 GCDC and porcine bile with βG and AX from various sources and figures of 13C NMR 506 spectra and SAXS pattern for βG and AX in the presence and absence of BS. 507 508 References 509 Anderson, J. W., Deakins, D. A., Floore, T. L., Smith, B. M., & Whitis, S. E. (1990). Dietary 510 fiber and coronary heart-disease. Critical Reviews in Food Science and Nutrition, 511 29(2), 95-147. 512 Belobrajdic, D. P., Bird, A. R., Conlon, M. A., Williams, B. A., Kang, S., McSweeney, C. S., 513 Topping, D. L. (2012). An arabinoxylan-rich fraction from wheat enhances caecal 514 fermentation and protects colonocyte DNA against diet-induced damage in pigs. 515 British Journal of Nutrition, 107(9), 1274-1282. 516 Bengtsson, S., Aman, P., Graham, H., Newman, C. 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Summary of 13C NMR effects observed for mixtures of SDF and GCDC (or 632 porcine bile (PB)) micelles. 633 Figures captions 634 Fig. 2. Molecular structure of the acids of major bile salts found in humans and 635 pigs. 636 Fig. 2. 13C NMR titration of 20 mM GCDC against (A) LMW barley βG showing 637 downfield chemical shift changes in GCDC resonances at 0-32.6 ppm; & (B) oat βG showing 638 changes in GCDC resonances at 0-38.0 ppm 639 Fig. 3. 13C NMR spectra of (1) 20 mM GCDC in the presence of (2) 1.67% LMW wheat & 640 (3) 1.67% rye AX showing changes in GCDC resonances at 0-38.0 ppm. 641 642 Fig. 4. Regions of 13C NMR spectra for porcine bile (spiked with TCDC, lower 643 traces) in the presence of (upper traces) 1.67% w/v MMW barley βG (A & B) & MMW 644 wheat AX (C & D) showing changes in BS resonance 645 Fig. 5. SAXS pattern for SDF in the presence and absence of BS: (top) MMW barley 646 βG in the presence and absence of GCDC; (bottom) MMW wheat AX in the presence and 647 absence of GCDC. ITotal is theoretical total intensity of SDF and BS when they are present in 648 solution in the absence of structural changes or inter-particle interactions. 649 650 651 652 21 H3C O 22 21 R’ 20 H3C O 18 22 CH R’ 20 3 24 25 18 23 CH H NH 3 24 25 12 23 17 SO3H H 19 11 13 12 NH 26 17 CH3 16 19 11 13 COOH 14 26 CH3 16 1 9 15 14 10 8 15 2 1 9 H 2 10 8 3 5 7 H 4 6 3 5 7 HO R 4 6 HO R H R” H R” R R’ R” R R’ R” OH OH H TCA OH OH H GCA H OH H TDCA H OH H GDCA OH H H TCDCA OH H H GCDCA OH H OH THCA OH H OH GHCA H H OH THDCA H H OH GHDCA Glycine conjugates of bile acids Taurine conjugates of bile acids Fig. 1. Molecular structure of the acids of major bile salts found in humans and pigs. B A Fig. 2. 13C NMR titration of 20 mM GCDC against (A) LMW barley βG showing downfield chemical shift changes in GCDC resonances at 0-32.6 ppm; & (B) oat βG showing changes in GCDC resonances at 0-38.0 ppm Fig. 3. 13C NMR spectra of (1) 20 mM GCDC in the presence of (2) 1.67% LMW wheat & (3) 1.67% rye AX showing changes in GCDC resonances at 0-38.0 ppm. 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 Fig. 4. Regions of 13C NMR spectra for porcine bile (spiked with TCDC, lower traces) in the presence of (upper traces) 1.67% w/v MMW barley βG (A & B) & MMW wheat AX (C & D) showing changes in BS resonance Fig. 5. SAXS pattern for SDF in the presence and absence of BS: (top) MMW barley G in the presence and absence of GCDC; (bottom) MMW wheat AX in the presence and absence of GCDC. ITotal is theoretical total intensity of SDF and BS when they are present in solution in the absence of structural changes or inter-particle interactions. 737 Table 1. Summary of 13C NMR effects observed for mixtures of SDF and GCDC (or 738 porcine bile (PB)) micelles. Spectral changes in BS SDF resonances βG AX Changes in chemical shifts: (a) downfield for low ppm; LMW, HMW barley βG Rye AX & MMW upfield for high ppm & MMW barley βG (PB) wheat AX (PB) (b) upfield for low ppm; Oat βG LMW & HMW wheat downfield for high ppm AX (c) small or no shifts MMW barley βG MMW wheat AX Peak height and/or area effects: (a) decrease Oat βG & HMW barley LMW, MMW & βG HMW wheat AX (b) small or no effect LMW & MMW barley Rye AX βG (c) inconclusive due to MMW barley βG (PB) MMW wheat AX (PB) overlapping resonances 739 740 741 742 33 743 Highlights 744 745 • NMR shows dynamic molecular interactions between bile salts and cereal soluble 746 fibres 747 • Chemical shift changes due to concentration or dilution of bile in presence of fibres 748 • Line broadening of bile resonances in presence of some fibres suggesting entrapment 749 • Bile salt induced conformation changes for β-glucan but not arabinoxylan from SAXS 750 751 34