Monosaccharides

The FFQ, originally developed for the TLGS, was a Willett-format questionnaire modified based on Iranian food items²⁵ and contains questions about average consumption and frequency for 168 food items during the past year.⁷ The food items were chosen according to the most frequently consumed items in the national food consumption survey in Iran.²⁵ Because different recipes are used for food preparation, the FFQ was based on food items rather than dishes, eg, beans, different meats and oils, and rice. Subjects indicated their food consumption frequencies on a daily basis (eg, for bread), weekly basis (eg, for rice and meat), monthly basis (eg, for fish), yearly basis (eg, for organ meats), or a never/seldom basis according to portion sizes that were provided in the FFQ. For each food item on the FFQ, a portion size was specified using USDA serving sizes (eg, bread, 1 slice; apple, 1 medium; dairy, 1 cup) whenever possible; if this was not possible, household measures (eg, beans, 1 tablespoon; chicken meat, 1 leg, breast, or wing; rice, 1 large, medium, or small plate) were chosen. Table 1shows food items and portion sizes used in the FFQ. Trained dietary interviewers with at least 3 of experience in the Nationwide Food Consumption Survey project²⁵ or TLGS^{26 (link)} administered the FFQs and 24-hour DRs during face-to-face interviews. The interviewer read out the food items on the FFQ, and recorded their serving size and frequency. The interview session took about 45 minutes. The interviewer for FFQ1 and FFQ2 was the same for each participant. Daily intakes of each food item were determined based on the consumption frequency multiplied by the portion size or household measure for each food item.²⁷ The weight of seasonal foods, like some fruits, was estimated according to the number of seasons when each food was available.
Dietary data were also collected monthly by means of twelve 24-hour DRs that lasted for 20 minutes on average. For all subjects, 2 formal weekend day (Thursday and Friday in Iran) and 10 weekdays were recalled. All recall interviews were performed at subjects’ homes to better estimate the commonly used household measures and to limit the number of missing subjects. Detailed information about food preparation methods and recipe ingredients were considered by interviewers. To prevent subjects from intentionally altering their regular diets, participants were informed of the recall meetings with dietitians during the evening before the interview. All recalls were checked by investigators, and ambiguities were resolved with the subjects. Mixed dishes in 24-hour DRs were converted into their ingredients according to the subjects’ report on the amount of the food item consumed, thus taking into account variations in meal preparation recipes. For instance, broth or soup ingredients—usually vegetables (carrot or green beans), noodles, barley, etc.—differed according to subjects’ meal preparation. Because the only available Iranian food composition table (FCT)²⁸ analyzes a very limited number of raw food items and nutrients, we used the USDA FCT²⁹ as the main FCT; the Iranian FCT was used as an alternative for traditional Iranian food items, like kashk, which are not included in the USDA FCT.
The food items on the FFQ and DR were grouped according to their nutrient contents, based on other studies,^{30 (link)} and modified according to our dietary patterns. Seventeen food groups were thus obtained, as follows: 1) whole grains, 2) refined grains, 3) potatoes, 4) dairy products, 5) vegetables, 6) fruits, 7) legumes, 8) meats, 9) nuts and seeds, 10) solid fat, 11) liquid oil, 12) tea and coffee, 13) salty snacks, 14) simple sugars, 15) honey and jams, 16) soft drinks, and 17) desserts and snacks (Table 1). The 168 food items on the FFQ were allocated to these 17 food groups, and the amounts in grams of each item were summed to obtain the daily intake of each food group.

Molecular weight distributions of lyophilized crude EPS were determined by size exclusion chromatography. In brief, crude EPS powder was suspended in 0.1 M NaNO₃ (0.5 mg/mL) and then filtered through a 0.45 μm pore diameter polyvinylidene fluoride membrane (Millipore Corporation, USA). The average molecular weight (MW) was determined by high-performance molecular exclusion chromatography (HPLC-SEC, Agilent 1,100 Series System, Hewlett-Packard, Germany) associated with a refractive index (IR) detector (Ibarburu et al., 2015 (link)). 50 μL of the samples were injected and eluted at a flow rate of 0.95 mL/min (pressure: 120:130 psi) at room temperature using 0.1 M NaNO₃ as mobile phase. Dextrans (0.5 mg/mL) with a molecular weight between 10³ and 2.10⁶ Da (Sigma-Aldrich, USA) were used as standards.
Once the molecular weight distributions were determined, low and high molecular weight fractions that composed the crude EPS obtained at 20°C were separated. For this purpose, EPS solutions (0.2% w/v) were centrifuged through a Vivaspin™ ultrafiltration spin column 100 KDa MWCO, (Sartorious, Goettingen, Germany) for 20 min at 6000 g, eluting only the low MW fraction. Subsequently, high MW fraction retained in the column was eluted using hot distilled water. The eluted fractions were passed through a Vivaspin column (cut-off 30KDa) in order to separate the middle and low MW fraction of EPS.
Monosaccharide composition of crude EPS and their fractions were determined by gas chromatography as previously described (Notararigo et al., 2013 (link)). Briefly, 1–2 mg of EPS were hydrolyzed in 1 mL of 3 M trifluoroacetic acid (1 h at 120°C). The monosaccharides obtained were converted into alditol acetates by reduction with NaBH₄ and subsequent acetylation. The samples were analyzed by gas chromatography in an Agilent 7890A coupled to a 5975C mass detector, using an HP5-MS column with helium as carrier gas at a flow rate of 1 mL/min. For each run, 1 μL of sample was injected (with a Split 1:50) and the following temperature program was performed: the oven was heat to 175°C for 1 min; the temperature was increased to 215°C at a rate of 2.5°C/min and then increased to 225°C at 10°C/min, keeping it constant at this temperature for 1.5 min. Monosaccharides were identified by comparison of retention times with standards (arabinose, xylose, rhamnose, galactose, glucose, mannose, glucosamine and galactosamine) analyzed under the same conditions. Calibration curves were also processed for monosaccharide quantification. Myo-inositol was added to each sample as internal standard.

BEAS-2B cells were grown in 12-well plates and exposed (3 wells per plate each) to (a) media alone, (b) 200 μM FAC, (c) 1000 μg/mL NAN (the predominant sialic acid in human cells and respiratory secretions), 1000 μg/mL sodium alginate (a polymer composed of mannuronate and guluronate monosaccharides), 1000 μM sodium guluronate (a uronate), or 1000 μM sodium hyaluronate (a polymer of disaccharides composed of glucuronate and N-acetyl-d-glucosamine) and (d) both 200 μM FAC and 1000 μg/mL NAN, 1000 μg/mL sodium alginate, 1000 μM sodium guluronate, or 1000 μM sodium hyaluronate. After 24 h incubation, the cells were gently washed, scraped into 10% trichloroacetic acid dissolved in 1.0 mL of 3 N HCl, digested at 70 °C, and non-heme iron concentrations were determined using ICPOES operated at a wavelength of 238.204 nm. Exposures of the BEAS-2B cells were repeated to (a) media alone, (b) 200 μM FAC, (c) 1000 μg/mL sodium alginate, and (d) both 200 μM FAC and sodium alginate for 24 h, the media was removed, cells were scraped into 0.5 mL DPBS and disrupted, and the ferritin concentrations quantified using an immunoturbidimetric assay.