Ninety hypercholesterolemic subjects were recruited for the study via flyers posted on the University of Minnesota campus and newspaper advertisements in the University of Minnesota Daily newspaper. To be included in the study, subjects needed to be healthy, non-smoking men and women between the ages of 22 and 65 years at risk for CVD (as defined by a total cholesterol greater than 200 mg/dl), with spoken and written English literacy. Subjects exclusion criteria included BMI > 30 upon admission to study; CVD, Diabetes Mellitus (fasting blood sugar > 126 mg/dl), chronic inflammatory diseases (e.g. Crohn's, rheumatoid arthritis), cancer in prior 5 years, renal or hepatic disease, recent bacterial infection (< 2 weeks), acute febrile illness in prior 2 months, history of drug or alcohol abuse in prior 6 months, lipid-lowering, anti-hypertensive or anti-inflammatory steroid medication use, active weight loss > 5 kg in prior 3 months (intended or unintended), concurrent or recent (within 30 days) intervention study participation. The inclusion and exclusion criteria were assessed via telephone screening, a health history questionnaire, and the initial total cholesterol screening visit. Subjects received oral and written information about the study, including a comprehensive study newsletter and individual written consent was obtained from each subject.
Subjects were asked to consume their usual diet with the addition of either a placebo supplement or dietary fiber from oat. The risks in the study were minimal. Subjects maintained their usual activities during the study with periodic visits for information sessions, sample collections, and supplement pickup.
The University of Minnesota Institutional Review Board Human Subjects Committee approved all aspects of this research. The study was a randomized, double-blind parallel group design. A total of 90 patients were enrolled with 45 patients per treatment arm. Subjects were randomly assigned to either placebo or treatment, stratified by age and sex. Fifteen subjects (n = 10 treatment, n = 5 placebo) were excluded from final analysis because their baseline cholesterol value was below 200 mg/dl despite a screening value above 200 mg/dl. Treatment was 6 grams of concentrated β-glucan from oat per day (from 12 g oat bran concentrate containing 54% oat β glucan). Placebo was 6 grams of dextrose monohydrate per day. Both were delivered as dietary supplements in the form of a powder. Subjects mixed their supplement with a beverage using a study-provided electric hand blender (Braun). Subjects were instructed to take the supplement via oral administration with their morning and evening meal for six weeks.
Subjects attended an initial pre-screening visit to verify eligibility for the study (visit 1). Subjects who met study criteria were scheduled for a baseline visit, one day before the intervention began (visit 2). Visits 3 and 4 were scheduled for day 21 (midpoint) and day 42 (final) of the intervention.
Three-day diet records were obtained from each subject at the midpoint and final visits. Subjects recorded two weekdays and one weekend day of their total food intake. All records were analyzed for total nutrient composition of each day's intake using the First DataBank Nutritionist Five software. Total calories, carbohydrate, fat, protein, cholesterol, saturated fat, monounsaturated fat, polyunsaturated fat, total fiber, soluble fiber, insoluble fiber and crude fiber were analyzed for each subject for average intake. Analysis of fiber did not include the addition of oat β-glucan to the diet.
At each visit subjects were asked to fast overnight for a minimum of 10 hours and then have blood samples drawn at the General Clinical Research Center, University of Minnesota, Twin Cities. Blood was drawn for the following measurements: plasma total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, apolipoprotein A-I, apolipoprotein B, complete blood count and differential, homocysteine, c-reactive protein and comprehensive metabolic panel. Plasma LDL cholesterol concentration was calculated using the Friedewald equation. All specimens collected were transported via courier to Quest Diagnostics (Wood Dale, IL) for analysis.
Blood pressure was measured at each visit for each subject using a standardized sphygmomanometer (Tycos). Three measurements were taken, one minute apart, with the subject in a semi-reclined position following the approved National Institutes of Health method . These three values were averaged individually for diastolic and systolic pressures. Standing weight was recorded every visit to the nearest 1/4 lb with light clothing and no shoes on a calibrated balance beam scale.
Each visit participants completed a short survey rating gastrointestinal effects on a scale related to normal. Symptoms recorded include: frequency of stools, consistency of stools, degree of intestinal bloating and degree of flatulence.
Model intestinal fermentation
Concentrated oat β-glucan (54% β-glucan), partially hydrolyzed guar gum (MW 400 kDa), and inulin (90% dp = 10, 10% dp = 1–2) were subjected to model intestinal fermentation . Glucose served as a positive control because it is fully fermentable by colonic bacteria. No fiber was the negative control to assess the concentrations of SCFA produced from substrates residing in the fecal inoculum. Chemical reagents were obtained from Fisher Scientific (New Hampton, NH, USA), Sigma Aldrich (St. Louis, MO, USA), and VWR Scientific (West Chester, PA, USA).
Briefly, the fibers were hydrated for 12 hours in 40 ml sterile trypticase peptone media fortified with minerals, phosphate buffer, and a reducing solution (950 ml distilled water, 6.25 g cysteine hydrochloride, 40 ml 1N NaOH, 6.25 g sodium sulfide monohydrate) at 4°C. Five-100 mL serum bottles were prepared for each fiber sample (0.5 g), one for each of the five time points: 0, 4, 8, 12, and 24 hours. Control bottles containing either 0.5 g glucose or no added carbohydrate were prepared in the same manner. Two hours prior to inoculation with fecal solution, sample bottles were warmed to 37°C. Fecal inoculum was prepared as described by McBurney and Thompson . Fecal samples from three human subjects consuming a nonspecified Western diet were pooled (125 g total) and diluted with 400 ml distilled water. The solution was homogenized in a blender. Reducing solution was added to the fecal inoculum to obtain a ratio of 15 parts fecal inoculum to 2 parts reducing solution .
Ten milliliters of fecal inoculum was added into each serum bottle along with 0.8 mL Oxyrase® oxygen reducing enzyme (Oxyrase Inc., Mansfield, OH). The bottles were immediately flushed with carbon dioxide gas to eliminate oxygen and generate anaerobic conditions. The bottles were gently shaken in a 37°C water bath. One sample bottle for each fiber was removed at 0, 4, 8, 12, and 24 hours. Immediately upon removal, 1 mL of copper sulfate (200 g/L) was added to each bottle to kill the bacteria and cease fermentation. Two-2 ml aliquots were removed. Samples were prepared for gas chromatography as described previously [16, 17].
Lactate, acetate, propionate, butyrate isobutyrate, 2-methylbutyrate, isovalerate, and valerate were determined by gas chromatography using a Hewlett Packard 6890 gas chromatograph (Hewlett Packard, Palo Alto, CA) with a 4% carbowax 20 M/80/120 carbopack B-DA column (Supleco, Bellefonte, PA) a temperature of 175 degrees C. Flow rates for nitrogen, hydrogen, and air were 24, 40, and 450 mL/min, respectively. All SCFA concentrations were corrected for the control concentration of SCFA at each time point. Molar ratios of SCFA were determined by dividing the number of moles of each SCFA (acetate, propionate, and butyrate) by the total moles of acetate, propionate, and butyrate.
Data was reported as means ± SEM. Changes were calculated by subtracting baseline from the 6-week value, so that positive changes indicate an increase. Baseline values and changes were compared between oat β-glucan and control by two-sample t-tests, while paired t-tests were used for within-group changes from baseline. In the model intestinal fermentation, all SCFA concentrations were corrected for the negative control concentration of SCFA at each time point. SCFA concentrations were compared between fibers at each time point separately, using Tukey pairwise procedure for multiple comparisons. Statistical analyses were performed with SAS statistical software package, versions 8.0 and 9.1 (SAS Institute Inc, Cary, NC, USA).