Nutrient Metabolism

Nutrient Metabolism ␤-Glucan Fractions from Barley and Oats Are Similarly Antiatherogenic in Hypercholesterolemic Syrian Golden Hamsters1 Bryan Delaney, Robert J. Nicolosi,*2 Thomas A. Wilson,* Ting Carlson,† Scott Frazer, Guo-Hua Zheng,† Richard Hess, Karen Ostergren, James Haworth and Nathan Knutson Cargill Health and Food Technologies, Wayzata, MN 55391; *Center for Health and Disease Research, Department of Health and Clinical Sciences, University of Massachusetts-Lowell, Lowell, MA 01854; and †Cargill Sweeteners North America, Dayton, OH 45414-4321 ABSTRACT The cholesterol-lowering activities of oats and barley are commonly attributed to the ␤-glucan fractions. Although ␤-glucan is present in both grains and appears to be chemically similar, the effect of source on cholesterol-lowering activity has not been evaluated. In the present study, the antiatherogenic properties of ␤-glucan concentrates from oats and barley were evaluated in Syrian golden F1B hamsters consuming a semipurified hypercholesterolemic diet (HCD) containing cholesterol (0.15 g/100 g), hydrogenated coconut oil (20 g/100 g) and cellulose (15 g/100 g). After a 2-wk lead-in period, control hamsters were fed the HCD, whereas experimental hamsters consumed HCD formulated to include ␤-glucan (2, 4, or 8 g/100 g) by addition of ␤-glucan concentrate prepared from oats or barley at the expense of cellulose. Compared with control hamsters, dosedependent decreases that were similar in magnitude in plasma total and LDL cholesterol concentrations were observed in hamsters fed ␤-glucan from either source at wk 3, 6 and 9. Compared with controls, liver cholesterol concentrations were also reduced (P ⬍ 0.05) in hamsters consuming 8 g/100 g oat or barley ␤-glucan. In agreement with previously proposed mechanisms, total fecal neutral sterol concentrations were significantly increased (P ⬍ 0.05) in hamsters consuming 8 g/100 g barley or oat ␤-glucan. Aortic cholesterol ester concentrations were significantly reduced (P ⬍ 0.05) in hamsters fed 8 g/100 g ␤-glucan from barley or oats. Although aortic total cholesterol and cholesterol ester concentrations were significantly correlated with LDL cholesterol (r ⫽ 0.565, P ⬍ 0.004 and r ⫽ 0.706, P ⬍ 0.0001, respectively), this association could explain only half of the variability. This study demonstrated that the cholesterol-lowering potency of ␤-glucan is approximately identical whether its origin was oats or barley. J. Nutr. 133: 468 – 495, 2003. KEY WORDS: ● ␤-glucan ● early atherosclerosis

●

LDL cholesterol

●

fecal neutral sterol

●

aortic cholesterol ester

Such preparations have demonstrated cholesterol-lowering activity in hypercholesterolemic hamsters (1–3,17) and humans (16,18). Although fewer clinical studies exist, barley foods also lower serum cholesterol concentrations in humans (19 –23). As with oats, domestic barley cultivars contain ␤-glucan, although at higher concentrations than in oats (15,24). However, although barley cultivars with the highest concentrations of ␤-glucan contain approximately twice the amount found in oats, consumption of large amounts of barley foods is also likely to be necessary for clinically relevant reductions in serum cholesterol concentrations. As with oats, concentrated ␤-glucan preparations from barley lower serum cholesterol concentrations in animal models of hypercholesterolemia including hamsters (2,25,26) and rats (27). Modest differences may exist, but the ␤-glucan in oats and barley appears to be similar structurally (28). It is therefore likely that ␤-glucan concentrates from either source would possess similar cholesterol-lowering activities. The present study was designed to compare the effects of concentrated ␤-glucan from oats and barley on plasma lipids and lipoprotein

Consumption of oats or oat bran lowers serum cholesterol concentrations in animal models of hypercholesterolemia (1– 5). The relevance of these studies to humans has been confirmed by numerous clinical studies demonstrating cholesterollowering activity in individuals with elevated serum cholesterol concentrations after consumption of oats (6,7) or oat bran [(8 –11); reviewed in (12)]. This activity is largely attributable to the soluble fiber fraction of oats, in particular to the (133,134)-␤-D-glucan (␤-glucan) component. Because the actual concentration of ␤-glucan in oats is relatively small [⬍5%; 13–15], clinically relevant reductions in serum cholesterol require the consumption of large amounts of whole-grain foods (16). Alternatively, ␤-glucan concentrates have been prepared from oats to enable oral consumption of ␤-glucan in amounts likely to be associated with health benefits while decreasing the need to consume large amounts of whole grains.

1 This study was sponsored by Cargill, Inc., Health and Food Technologies, Wayzata, MN. 2 To whom correspondence should be addressed. E-mail: [email protected].

0022-3166/03 $3.00 © 2003 American Society for Nutritional Sciences. Manuscript received 28 August 2002. Initial review completed 25 September 2002. Revision accepted 5 November 2002. 468

CHOLESTEROL LOWERING BY BARLEY AND OAT ␤-GLUCAN

and hepatic cholesterol, fecal excretion of neutral sterols and total bile acids, and antiatherogenic properties in hamsters consuming a hypercholesterolemic diet (HCD)3. Hamsters were selected for this study because decreased plasma cholesterol concentrations have been reported in hamsters fed HCD after consumption of ␤-glucan– enriched fractions from oats and barley (1– 4,17,25). Furthermore, hamsters represent a useful model for humans because they metabolize cholesterol similarly (29,30) and their lipoprotein profile resembles that of humans fed HCD (2). The relevance of the hamster model to humans has been established by the consistent ability of hamsters to respond to dietary modulation of cholesterol absorption similarly to humans (2,30). MATERIALS AND METHODS Preparation of ␤-glucan concentrate from barley and oats. ␤-Glucan concentrate (Barley Betafiber, Cargill, Inc., Wayzata, MN) was prepared from hulless barley (Azhul variety) by an extraction process similar to that of Aman and Hesselman (31) with slight modifications. Ground barley flour was mixed with boiling water and bacterial ␣-amylase to extensively hydrolyze starch, solubilize ␤-glucan and inactivate contaminating ␤-glucanase activities. Solubilized ␤-glucan in the clarified liquid fraction was precipitated, washed with ethanol and dried. A similar procedure was used for the extraction of ␤-glucan from oat bran (SCM 350 oat bran from ConAgra Foods, S. Sioux City, NE) except protease (papain) was used to hydrolyze protein in addition to the starch hydrolysis step and hexane was used to extract excess oil from dried oat ␤-glucan concentrate. Proximate analysis of the concentrated ␤-glucan preparations is presented in Table 1. Diets. All diets were formulated and dietary ingredients supplied by Research Diets (New Brunswick, NJ) except for barley and oat ␤-glucan concentrates, which were prepared by Cargill, Health and Food Technologies (Wayzata, MN). Diet ingredients for all groups were identical except for the nutrient under evaluation as indicated in Table 2. The control HCD contained 15 g/100 g cellulose, whereas the experimental diets were formulated to contain 2, 4 or 8 g/100 g ␤-glucan from oats or barley by the addition of the ␤-glucan concentrate from the respective source at the expense of cellulose. Analytical characterization of ␤-glucan was conducted on hamster feed to validate the composition and homogeneity of dietary blending and stability under conditions of storage and use. Hamster feed was milled and ␤-glucan was quantified using a Megazyme (Megazyme International Ireland, Bray, Wicklow, Ireland) kit version of the McCleary method [AOAC method 995.16; see (32,33)]. Animals. Male Syrian golden hamsters (n ⫽ 70; F1B strain, BioBreeders, Fitchburg, MA) ⬃8 –10 wk old were acclimated to individual stainless steel cages and fed HCD for 2 wk before initiation of the experimental diets. Hamsters were then bled to determine plasma concentrations of total (TC), HDL (HDL-C) and LDL cholesterol (LDL-C); they were divided into 7 groups (n ⫽ 10/group) with similar starting plasma LDL-C concentrations and fed the specified diets. The experimental protocols were approved by the Institutional Animal Care and Use Committee. Hamsters were maintained in accordance with guidelines of the Animal Care Committee at the University of Massachusetts-Lowell Research Foundation and the NIH. Hamsters were housed in environmentally controlled conditions with an alternating 12-h light:dark cycle and consumed food and water ad libitum except when food was withheld for the experimental protocols described below. Plasma lipoprotein cholesterol and triglyceride measurements. Blood was collected via the retro-orbital sinus into heparinized tubes from hamsters deprived of food for 12 h. Plasma was harvested after centrifugation at 1500 ⫻ g at room temperature for 10 min, and plasma TC (34) and triglyceride (TG) (35) concentrations were measured enzymatically. Plasma LDL-C (combination of VLDL, in-

3 Abbreviations used: GC, gas chromatography; HCD, hypercholesterolemic diet; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; TC, total cholesterol; TG, triglyceride.

469

TABLE 1 Proximate composition of ␤-glucan concentrates from barley and oats %

␤-Glucan1 Hydrolyzed starch2 Protein3 Moisture4 Oil5 Ash6

Barley

Oats

77.98 6.53 7.85 5.20 0.96 1.82

64.88 8.66 11.10 8.80 1.64 2.78

1 ␤-Glucan concentration was determined by AOAC method 995.16 using Megazyme mixed-linkage ␤-glucan assay kit (32, 33). 2 Hydrolyzed starch was determined enzymatically using amyloglucosidase. 3 Protein concentration was determined by AOAC method 991.20 (32). 4 Moisture was determined by AOAC method 926.08 (32). 5 Oil concentration was determined by AOAC method 933.05 (32). 6 Ash concentration was determined using Corn Refiners Association Standard Analytical Method A-4 (62).

termediate, and LDL-C) was precipitated with phosphotungstate reagent (36) and HDL-C was measured in the supernatant. The concentration of LDL-C was calculated as the difference between plasma TC and HDL-C. The accuracy and precision of the procedures used for the measurements of plasma TC and HDL-C were maintained by participation in the Lipid Standardization Program of the Centers for Disease Control and the National Heart, Blood and Lung Institute (Atlanta, GA). All reagents used were supplied by SigmaAldrich (St. Louis, MO). All assays were performed using a Cobas Mira Plus (Roche Diagnostic Systems, Basel, Switzerland) clinical chemistry autoanalyzer using 10 ␮L of plasma for each sample (Roche Pharmaceuticals). Aortic cholesterol measurements. At the end of the exposure period (wk 9), hamsters were anesthetized with an intraperitoneal injection of sodium pentobarbital and aortic tissue was obtained for determination of cholesterol concentration. The heart and thoracic aorta were removed and stored in PBS at 4°C for subsequent analysis. To measure cholesterol concentrations in the aortic arch, a piece of aortic tissue extending from as close to the heart as possible to the branch of the left subclavian artery was used (⬃20 – 40 mg). The tissue was cleaned, weighed and placed in a vial containing 4 mL of methanol and 10 mL of chloroform and treated as described by Rudel et al. (37). The sample was mixed vigorously and left at room temperature for 48 h before extraction. The solution was then placed in a 37°C water bath, under N2. When one half of the solution was evaporated, 1 mL of chloroform with 1% Triton-100 was added, mixed and evaporated to dryness at 37°C under N2. Distilled water (250 ␮L) was added to the samples, vortexed and placed in a shaking water bath at 37°C for 20 min to solubilize the lipid. After incubation, aortic total and free cholesterol concentrations were determined enzymatically in triplicate with 25 ␮L of sample (Wako Chemicals, Richmond, VA) using an ELISA assay. The aortic cholesteryl ester concentration was calculated as the difference between the total and the free cholesterol concentrations. A pilot study was conducted to evaluate the extent to which this procedure removed tissue cholesterol. Aortic cholesterol concentrations were determined after tissue was placed in solvent (4 mL of methanol and 10 mL of chloroform) overnight with frequent vigorous mixing and compared with the concentrations obtained after tissue minced or homogenization. No significant differences in aortic free or cholesterol ester content were observed between the different cholesterol extraction procedures with efficiencies of ⬃96%. Hepatic cholesterol measurements. Hepatic cholesterol concentrations were measured by a previously described method (38) in the following manner: a 100-mg portion of liver was homogenized with

DELANEY ET AL.

470

TABLE 2 Diet composition Barley ␤-glucan (g/100 g) Control

2

4

Oat ␤-glucan (g/100 g) 8

2

4

8

g/kg Casein, 80 mesh DL-Methionine MaltoDextrin 10 Cornstarch Sucrose Cellulose BW200 Barley ␤-glucan concentrate1 Oat ␤-glucan concentrate Safflower oil Coconut oil, hydrogenated Mineral mix, S100012 Vitamin mix, V100013 Choline bitartrate Cholesterol Yellow dye, FD&C#5 Red dye, FD&C#40 Blue dye, FD&C#1

250 5 125 156 95 150 0 0 20 200 35 10 2 1.5 0 0 0

250 5 125 156 95 121.5 28.5 0 20 200 35 10 2 1.5 0.1 0 0

250 5 125 156 95 93 57 0 20 200 35 10 2 1.5 0 0.1 0

250 5 125 156 95 36 114 0 20 200 35 10 2 1.5 0 0 0.1

250 5 125 156 95 116.75 0 33.25 20 200 35 10 2 1.5 0.05 0.05 0

250 5 125 156 95 83.5 0 66.5 20 200 35 10 2 1.5 0.05 0 0.05

250 5 125 156 95 17 0 133 20 200 35 10 2 1.5 0 0.05 0.05

1 Barley Betafiber, Cargill, Inc., Wayzata, MN 2 Mineral mix, S10001 composition (g/kg): calcium carbonate, 160.0; calcium phosphate, 235.0; magnesium oxide, 5.0 g; potassium citrate, 420.0

g; potassium sulfate, 20.0; sodium chloride, 66.7; chromium potassium sulfate 0.1; cupric carbonate, 0.14; potassium iodate, 0.02; ferric citrate, 4.0; manganous carbonate, 1.4; sodium selenite, 0.003; zinc carbonate, 0.75; sucrose, 46.887. 3 Vitamin mix, V10001 composition (amount in 10 grams): vitamin A palmitate, 20,000 IU; vitamin D-3, 1,000 IU; vitamin E acetate, 50 IU; menadione sodium bisulfate, 0.5 mg; biotin, 0.3 mg; cyanocobalamin, 10 ␮g; folic acid, 6 mg; nicotinic acid, 30 mg; calcium pantothenate, 30 mg; pyridoxine-HCl, 6 mg; riboflavin, 6 mg; thiamin-HCl, 6 mg; ascorbic acid, 500 mg.

50 mg of sodium sulfate. Methanol (5 mL) was then added and the tissue homogenized a second time followed by addition of 10 mL of chloroform. After mixing, 3 mL of a solution containing 1.25% KCl and 0.05% H2SO4 was added and centrifuged at 400 ⫻ g at room temperature for 10 min. The bottom layer was transferred and the supernatant reextracted with 3 mL of chloroform/methanol (2:1) and centrifuged at 400 ⫻ g at room temperature for 10 min. The bottom layer was transferred and pooled with the previous step. The solution was placed in a 37°C water-bath and placed under N2. When approximately half of the solution was evaporated, 1 mL of chloroform with 1% Triton-100 was added, mixed and evaporated to dryness at 37°C under N2. Distilled water (500 ␮L) was added to the samples, vortexed and placed in a shaking water bath at 37°C for 20 min to solubilize the lipid. After incubation, hepatic total and free cholesterol concentrations were determined enzymatically (Wako Chemicals) using an ELISA assay. Hepatic cholesteryl ester concentration was calculated as the difference between the total and the free cholesterol concentrations. Fecal neutral sterol measurements. Fecal samples were collected over the final 3 d of the exposure period, freeze-dried (lyophilized) and ground before analysis (39). Dry feces (200 mg) were extracted with 4 mL of methanol/chloroform (50:50) for 1 h at 100°C in a 5-mL Reacti-vial (Pierce, Rockford, IL) fitted with a mini-nert cap. Samples were then allowed to come to room temperature and were centrifuged at 500 ⫻ g at room temperature for 10 min. The supernatant was removed from the fecal pellet and transferred to an 8-mL borosilicate vial. Supernatants were evaporated to dryness at 50°C under N2. Then, 4 mL of 0.1 mol/L NaOH/ethanol (10:90, v/v) was added to each sample, overlaid with N2, capped and heated at 100°C for 30 min. The samples were allowed to cool to room temperature; the solvent was removed and transferred to 16 ⫻ 150 mm borosilicate test tubes. Water (5 mL) and 3 mL of hexane were added to the solvent followed by vortexing and centrifugation at 500 ⫻ g for 2 min. The top hexane layer was removed and placed in vials. The hexane extraction was repeated two more times and pooled. The hexane extracts were stored at ⫺80°C until analysis of neutral sterols. The aqueous layer was adjusted to 10 mL with the addition of 0.1

mol/L NaOH, and 1 mL was removed for quantitative enzymatic determination of total bile acids. To the hexane portion, 1 mL of 5-␣-cholestane (240 mg/L) (Sigma-Aldrich) was added and the solution was adjusted to 10 mL with hexane in a volumetric flask. After 2 mL were removed and evaporated to dryness at 100°C under N2, 100 ␮L of Tri-Sil reagent was added and the samples heated at 85°C for 20 min, followed by evaporation and reconstitution in 100 ␮L of methylene chloride. Then 1 ␮L was injected and analyzed by capillary gas chromatography (GC). GC analyses. Neutral sterols were analyzed using a Shimadzu (Kyoto, Japan) GC-14A gas chromatograph with a flame ionization detector and a 50 m ⫻ 0.2 mm HP-1 capillary column (Hewlett Packard, Andover, MA). The injector and detector temperatures were set at 300°C. The initial column temperature was 220°C and was increased to 300°C at a rate of 2°C/min. The final temperature was held for 10 min. Column flow rate was 1.5 mL/min. Peak areas were quantitated using a Shimadzu CR501 integrator. Quantification and identification of neutral sterols were based on the appropriate purified external standards supplied by Sigma-Aldrich. Extraction efficiency for neutral sterol sterols following this protocol was ⬃94%. Total fecal bile acid measurements. Using Sigma Procedure #450, blank and test reagents were prepared as described in assay instructions. Standard solutions were prepared using known quantities of lithocholic acid (Sigma-Aldrich). Standards and samples (16 ␮L of the 1 mL aqueous portion plus 24 ␮L of calf serum) were assayed in two sets of triplicates (one set for 100 ␮L of test reagent, the other set for 100 ␮L of blank reagent) on 96-well microtiter plate. After 10 min incubation at 37°C, absorbances were read at 530 nm using a Molecular Devices (Sunnyvale, CA) microplate reader. The extraction efficiency for bile acids from hamster feces following this protocol is ⬃92%. Statistical analysis. One-way ANOVA was used to examine the effect of treatment on the different variables using SigmaStat software (Jandel Scientific, San Rafael, CA). Plasma lipid and lipoprotein cholesterol concentrations in ␤-glucan–treated hamsters were compared with the HCD control group using Dunnett’s t test. Intergroup comparisons (e.g., 2 g/100 g oats vs. 2 g/100 g barley) were evaluated


471

TABLE 3 Plasma total cholesterol concentrations of hamsters consuming concentrated ␤-glucan from oats or barley1 ␤-Glucan concentrate

wk 0

wk 3

wk 6

wk 9

7.43 ⫾ 0.56a 7.75 ⫾ 0.63a 7.43 ⫾ 0.49a 6.24 ⫾ 0.70b,c 6.49 ⫾ 0.90b 5.52 ⫾ 0.61c 5.85 ⫾ 1.00c

7.60 ⫾ 0.86a 7.97 ⫾ 1.08a 6.97 ⫾ 1.02a 6.33 ⫾ 1.07b,c 6.53 ⫾ 0.98b 5.15 ⫾ 0.83c 5.58 ⫾ 1.09c

mmol/L 5.86 ⫾ 0.47a 6.02 ⫾ 0.69a 5.84 ⫾ 0.61a 5.72 ⫾ 0.54a,2 5.80 ⫾ 0.58a 6.09 ⫾ 0.84a 6.12 ⫾ 0.84a

0 (Control Oat, 2 g/100 g Barley, 2 g/100 g Oat, 4 g/100 g Barley, 4 g/100 g Oat, 8 g/100 g Barley, 8 g/100 g

7.61 ⫾ 1.13a 6.46 ⫾ 1.78a 7.51 ⫾ 1.08a,2 5.65 ⫾ 1.65b,c 6.06 ⫾ 1.32b,3 4.95 ⫾ 0.54c 5.24 ⫾ 0.83c

1 Values are means ⫾ SD, n ⫽ 10 except where noted. Means in each column with different superscript letters differ (P ⬍ 0.05). 2 n ⫽ 9. 3 n ⫽ 8.

using Student’s t test. Correlations between LDL-C and aortic TC and aortic cholesterol ester were determined using pooled samples of both 8 g/100 g ␤-glucan from barley and oats by Pearson’s Productmoment method as previously reported (40) and plotted using SigmaStat software (Jandel Scientific, San Rafael, CA).

RESULTS Isolation of ␤-glucan and dietary incorporation. Concentrations of ␤-glucan from the barley and oat preparations were 78 and 65%, respectively (Table 1). The ␤-glucan– enriched fraction of oats was prepared using a similar process but yielded 65% ␤-glucan in the oat extract (Table 1). Because the concentration of ␤-glucan from the barley concentrate was higher than that of the oat preparation, the addition of smaller amounts of the barley preparation was necessary to obtain the same dietary concentration of ␤-glucan when incorporated into the hamster feed. These fractions were then blended into a semisynthetic HCD at concentrations corresponding to 2, 4, or 8 g/100 g (Table 2). The ␤-glucan was incorporated homogeneously and was stable under the conditions used to blend, ship and store the experimental diets (data not shown). Plasma TC and TG concentrations. Plasma TC concentrations in hamsters consuming the HCD were approximately the same in all groups after a 2-wk lead-in period (Table 3). After that period, hamsters were fed the indicated diets. Compared with hamsters fed the HCD, a dose-dependent decrease in plasma TC concentrations was observed at all time points after consumption of the experimental diets containing 4 and 8 g/100 g ␤-glucan from oats or barley, but not in hamsters

consuming 2 g/100 g ␤-glucan from either source. The decrease in plasma TC concentrations was not distinguishable on the basis of the source of ␤-glucan. There were no effects on plasma TG concentrations, food intake and body weight in any of the treatment periods (data not shown). Plasma LDL-C concentrations. Similar to plasma TC concentrations, a dose-dependent decrease was observed in LDL-C concentrations in hamsters fed diets supplemented with 4 and 8 g/100 g ␤-glucan from either oats or barley (Table 4). However, at 2 g/100 g ␤-glucan from either source, no changes were observed compared with HCD control. Importantly, the plasma LDL-C concentrations in hamsters treated with the 4 and 8 g/100 g concentrations of ␤-glucan from either source were not different from each other at any time point. Plasma HDL-C concentrations. Plasma concentrations of HDL-C were similar in all groups after consumption of the HCD for 2 wk (Table 5). Compared with the HCD control group, consumption of 8 g/100 g ␤-glucan–supplemented diets from both sources caused decreases in plasma HDL-C concentrations at 6 and 9 wk. Surprisingly, a significant increase in plasma HDL-C concentrations compared with HCD control (P ⬍ 0.05) was observed in the 2 g/100 g oat group that did not occur in the barley ␤-glucan group at 9 wk. Liver cholesterol concentrations. After 9 wk, liver total, free and esterified cholesterol concentrations were significantly decreased in hamsters consuming 8 g/100 g ␤-glucan from oats or barley compared with the control group. Although the difference in liver TC was significant (P ⬍ 0.05) compared

TABLE 4 Plasma LDL cholesterol concentrations of hamsters consuming concentrated ␤-glucan from oats or barley1 ␤-Glucan concentrate

wk 0

wk 3

wk 6

wk 9

4.63 ⫾ 0.48a 4.80 ⫾ 0.42a 4.99 ⫾ 0.51a 3.84 ⫾ 0.53b 3.96 ⫾ 0.82b 3.28 ⫾ 0.52b 3.59 ⫾ 0.84b

5.07 ⫾ 0.76a 5.13 ⫾ 0.58a 4.45 ⫾ 1.01a 3.75 ⫾ 0.82c 4.08 ⫾ 0.89b 3.01 ⫾ 0.56c 3.53 ⫾ 0.93c

mmol/L 0 (Control) Oat, 2 g/100 g Barley, 2 g/100 g Oat, 4 g/100 g Barley, 4 g/100 g Oat, 8 g/100 g Barley, 8 g/100 g

3.77 ⫾ 0.47a,2 3.92 ⫾ 0.88a 3.64 ⫾ 0.67a 3.70 ⫾ 0.59a 3.68 ⫾ 0.63a,2 3.96 ⫾ 0.65a 3.89 ⫾ 0.88a

4.91 ⫾ 1.24a 4.16 ⫾ 1.96a 4.82 ⫾ 1.21a 3.17 ⫾ 1.70b,c 3.93 ⫾ 1.59a 2.74 ⫾ 0.44c 2.82 ⫾ 0.82c

1 Values are means ⫾ SD, n ⫽ 10 except where noted. Means in each column with different superscript letters differ (P ⬍ 0.05). 2 n ⫽ 9.

DELANEY ET AL.

472

TABLE 5 Plasma HDL cholesterol concentrations of hamsters consuming concentrated ␤-glucan from oats or barley1 ␤-Glucan concentrate

wk 0

wk 3

wk 6

wk 9

2.51 ⫾ 0.33a 2.77 ⫾ 0.31a 2.44 ⫾ 0.16a 2.41 ⫾ 0.01a 2.53 ⫾ 0.24a 2.24 ⫾ 0.20b 2.26 ⫾ 0.26b

2.53 ⫾ 0.31a 3.09 ⫾ 0.54b,2 2.53 ⫾ 0.26a 2.59 ⫾ 0.34a 2.45 ⫾ 0.46a 2.14 ⫾ 0.36b 2.05 ⫾ 0.28b

mmol/L 0 (Control) Oat, 2 g/100 g Barley, 2 g/100 g Oat, 4 g/100 g Barley, 4 g/100 g Oat, 8 g/100 g Barley, 8 g/100 g

2.29 ⫾ 0.59a 2.09 ⫾ 0.26a 2.20 ⫾ 0.14a 2.03 ⫾ 0.33a 2.22 ⫾ 0.43a 2.23 ⫾ 0.23a 2.06 ⫾ 0.26a,2

2.70 ⫾ 0.46a 2.31 ⫾ 0.54a 2.64 ⫾ 0.36a 2.42 ⫾ 0.16a,2 2.51 ⫾ 0.35a 2.47 ⫾ 0.14a 2.42 ⫾ 0.35a

1 Values are means ⫾ SD, n ⫽ 10 except where noted. Means in each column with different superscript letters differ (P ⬍ 0.05). 2 n ⫽ 9.

with hamsters fed the HCD, there were no significant differences between oat or barley glucan groups (Fig. 1). Hepatic cholesterol concentrations were not evaluated in hamsters consuming 2 or 4 g/100 g ␤-glucan from oats or barley. Fecal neutral sterol and total bile acid concentrations. Consumption of 8 g/100 g ␤-glucan from oats or barley significantly increased (P ⬍ 0.05) total fecal neutral sterol concentrations by 76 and 78%, respectively (Table 6) relative to the HCD. Virtually all individual sterols analyzed were greater for hamsters fed the barley and oat ␤-glucan compared with the HCD group, with the greatest quantitative increases in excretion observed for cholesterol (169 –218%) and coprostanol (43–52%). Although some differences were noted between fecal excretion of individual and total neutral sterols between the barley and oat groups, none were significant (P ⬎ 0.05). No significant differences were observed between any dietary treatments for fecal excretion of total bile acids (Table 6). Fecal neutral sterol and total fecal bile acid concentrations were not evaluated in hamsters consuming 2 or 4 g/100 g ␤-glucan from oats or barley. Aortic cholesterol concentrations. Compared with the HCD control group, a significant decrease in aortic TC con-

centration was observed in both the oat (⫺71%) and barley (⫺58%) groups (P ⬍ 0.05; Fig. 2). The difference in aortic TC concentration between the oat and barley groups was not significant (P ⫽ 0.206). Although the concentration of aortic free cholesterol was not different from the cholesterol control group in either of the ␤-glucan groups, the aortic cholesterol ester concentrations were profoundly decreased in hamsters fed 8 g/100 g ␤-glucan from oats (⫺84%) or barley (⫺80%). When data from both 8 g/100 g oat and barley ␤-glucan groups were pooled, the aortic TC and cholesterol ester concentrations were significantly correlated with LDL-C (r ⫽ 0.565, P ⬍ 0.004, r ⫽ 0.706, P ⬍ 0.0001, respectively). However, only 50% of the variability could be attributed to this association. Aortic cholesterol concentrations were not evaluated in hamsters consuming 2 or 4 g/100 g ␤-glucan from oats or barley. DISCUSSION The cholesterol-lowering activity of oats and barley is believed to be attributable to the ␤-glucan in the soluble fiber fraction of these cereal grains. ␤-Glucan is composed of highmolecular-weight water-soluble cell-wall polysaccharides consisting of (133,134)-␤-D-linked glucopyranosyl-monomers (41). The ␤-glucan in both grains is similar in structure; TABLE 6 Fecal neutral sterol and total bile acid concentrations of hamsters consuming concentrated ␤-glucan from oats or barley1 8 g/100 g ␤-Glucan Control

Oat

Barley

mg/g of feces

FIGURE 1 Concentrations of cholesterol (total, free, and esterified) in the livers of male Syrian golden F1B hamsters after 9 wk consumption of diets formulated with cellulose control, or 8 g/100 g ␤-glucan from concentrated oat or barley ␤-glucan. Data are expressed as means ⫾ SD, n ⫽ 10. Bars with different letters differ (P ⬍ 0.05).

Cholesterol Coprostanol Coprostanone Campesterol Stigmasterol Sitosterol Sitostanol Total neutral sterols Total fecal cholesterol Total fecal bile acids

1.34 ⫾ 0.9a 5.15 ⫾ 2.0a 0.06 ⫾ 0.1a 0.81 ⫾ 0.3a 0.11 ⫾ 0.1a 0.09 ⫾ 0.0a 0.09 ⫾ 0.1a 7.63 ⫾ 3.2a 6.54 ⫾ 2.8a 2.69 ⫾ 1.5a

4.24 ⫾ 2.9b 7.37 ⫾ 2.2b 0.05 ⫾ 0.1a 1.82 ⫾ 0.5b 0.24 ⫾ 0.1b 0.30 ⫾ 0.2b 0.25 ⫾ 0.2a 14.27 ⫾ 4.4b 11.51 ⫾ 4.2b 3.38 ⫾ 1.0a

3.59 ⫾ 2.8b 7.83 ⫾ 1.9b 0.10 ⫾ 0.2a 1.71 ⫾ 0.5b 0.16 ⫾ 0.1a 0.24 ⫾ 0.2a 0.25 ⫾ 0.2a 13.86 ⫾ 5.0b 11.66 ⫾ 3.6b 2.65 ⫾ 1.1a

1 Values represent means ⫾ SD, n ⫽ 10. Means in each row with different superscript letters differ (P ⬍ 0.05).


FIGURE 2 Concentrations of cholesterol (total, free, and esterified) in the aortas of male Syrian golden F1B hamsters after 9 wk consumption of diets formulated with cellulose control, or 8 g/100 g ␤-glucan obtained from concentrated oat or barley ␤-glucan. Data are expressed as mean ⫾ SD, n ⫽ 8/group because 2 outliers were detected in each group. Bars with different letters differ (P ⬍ 0.05).

however, differences with respect to the ratio of (133) to (134)-linkages (42,43), molecular weight (28) and possibly solubility (15) have been reported. Concentrated ␤-glucan from both sources lowers plasma cholesterol concentrations in clinical studies (16,18) and in animal models of hypercholesterolemia (1–3,17,25–27), but a comparison of the effects of ␤-glucan from different sources has not been reported. In the current study, ␤-glucan concentrates were prepared from oats and barley and their antiatherogenic properties evaluated in hypercholesterolemic hamsters. Consumption of semipurified diets containing concentrated oat or barley ␤-glucan indicated a dose-dependent decrease in plasma TC concentrations regardless of the source of the ␤-glucan concentrate. The magnitude of the decrease was consistent with previous studies evaluating the effect of ␤-glucan concentrates from barley (2,25) and similar to (3) or slightly greater in magnitude than the effect of oat bran and oat ␤-glucan concentrates in hamsters (1,2,17,44). This effect was primarily attributable to decreased plasma LDL-C concentrations. Decreased HDL-C concentrations were also observed in hamsters consuming high concentrations of ␤-glucan from oats and barley as has been reported in other studies evaluating the effect of barley or oat preparations in hamsters (2,317,25). The clinical relevance of the latter observation is questionable because it does not appear to occur in humans (6 – 8,10, 11,16,19,21). However, the results from this study demonstrate that the plasma cholesterol–lowering activity of ␤-glucan is approximately the same whether it is from barley or oats. Similar observations have been reported in rats consuming HCD supplemented with oat and barley gums (45). The ability of soluble fiber, and specific components therein, to lower serum cholesterol concentrations is believed to occur through a combination of mechanisms. Consumption of ␤-glucan inhibits absorption of cholesterol from the gut as demonstrated by a significant increase in the excretion of fecal cholesterol and neutral sterols (7,44,46). In the current study, a large increase in the fecal excretion of neutral sterols and cholesterol was observed after consumption of concentrated

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oat or barley ␤-glucan. Lia and co-workers (47) reported that oats caused a greater increase in sterol excretion than barley; however, there was no difference between oats and barley in the current report. This observation supports the concept that the cholesterol-lowering properties of ␤-glucan are attributable at least in part to inhibition of cholesterol absorption from the gut. Fibers containing ␤-glucan have also been reported to increase excretion of bile acids, suggesting a causative role in the lowering of plasma cholesterol concentrations (7,11,48). However, this association is not consistent because numerous studies have reported a cholesterol-lowering effect in the plasma without alteration of fecal bile acid concentrations (49 –52). Neither source of ␤-glucan concentrate altered fecal bile acid concentrations in the current study. Alternatively, consumption of ␤-glucan– containing fibers increases fecal output (7,48). Therefore, similar concentrations of bile acid relative to fecal weight may represent an increase in bulk excretion of bile acids; however, fecal output was not evaluated in the current study. Although many studies have shown that consumption of ␤-glucan decreases plasma lipid and lipoprotein cholesterol concentrations, the current study also demonstrated a striking decrease in the concentration of aortic cholesterol. This effect was attributable almost entirely to the cholesterol ester contents of the aorta, and again there was no difference in magnitude between oats and barley. This observation is particularly important because this form of tissue cholesterol is viewed as the hallmark in fatty streak formation. This is the first time that dietary ␤-glucan concentrates from any source have demonstrated such an effect on the aorta. Equally surprising was the finding that only 50% of the association could be explained by the effects of ␤-glucan on plasma LDL-C concentrations. This suggests that additional antiatherogenic mechanism(s) of action of ␤-glucan may occur outside of the gut. For example, some studies have reported a decrease in hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity after consumption of diets containing barley or oats (53). Alternatively, changes may also occur at the level of the blood vessel wall within the aorta. Oxidative stress (54) and inflammation (55) have been implicated in the development of early and advanced atherosclerosis. Therefore, dietary antioxidants could contribute to the effects observed in aortic cholesterol ester concentrations in the current study. For example, recent studies in guinea pigs have suggested that consumption of certain fibers may have antioxidant-like properties that are measurable in circulating LDL-C (56). Numerous antioxidants have been identified in both barley and oats (57–59) that may also have been concentrated in the process of preparing the ␤-glucan concentrates used in the current study. Further, substances not associated with the soluble fiber fractions have been identified in these grains and may contribute to the effect by inhibiting endogenous cholesterol production (60,61). However, it is not known whether the procedures used to concentrate the ␤-glucan fractions concentrate or dilute these substances and their concentrations were not evaluated in either ␤-glucan preparation used in this study. The results of the current study demonstrated that ␤-glucan concentrates from barley and oats modify plasma cholesterol concentrations and other indicators associated with atherogenic progression in hamsters with similar potency and through similar mechanisms of action. The effects were dependent on dietary concentrations of ␤-glucan but no differences were observed between the barley and oat preparations. Therefore, the structural differences between the ␤-glucan in barley and oats (15,28,42,43) may not be important with

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