Effects of Dietary Sulfur on Gut Microbiota H2S Production Merry Huang

T35 Medical Student Summer Research Program 08.24.16 Gut Microbiota Roles in Human Health

• Nutrient Metabolism

• Drug Metabolism

• Protection Against Pathogens

• Integrity of Gut Mucosal Barrier

• Immunomodulation Diet Contributes to Variations in Human Gut Microbiome

Lozupone, C. A Figure 5 | Human microbial diversity and enterotypes • Microbiome differences between western diet vs plant based diet • Characteristic changes in gut microbiome in disease states (e.g. obesity, inﬂammatory bowel disease) Global Prevalence of Inﬂammatory Bowel Disease

• Highest prevalence among industrialized countries with western diet Increased H2S Production in IBD – Modiﬁable via Diet

• Hydrogen Sulﬁde is toxic!

• Exogenous production from Sulfate Reducing Bacteria (SRB)

• Amino acids (cys, met) serve as primary sulfur substrates for SRB in sulfur gas production

Linden DR Can a dietary intervention of a low sulfur diet serve as a promising prescription for IBD management? Study Design Flow Chart Dietary Guidelines for Sulfur Diet Interventions High-S Diet1 Low-S Diet Meats: beef, pork, lamb, veal, venison, goat, processed meats (ham, bacon, bologna, salami, sausages, hot dogs, deli or lunch meats), poultry, Grains2: wheat, rice, corn, bulgur, buckwheat, barley, oats, breakfast cereals seafood, shellfish Dairy alternatives: almond milk, rice milk, hemp milk, coconut milk, cream Dairy Products: cheese, milk, yogurt, ice cream, sour cream substitutes Eggs Egg substitutes Plant-derived fats: oils (e.g., vegetable, olive, safflower, cottonseed, cod liver, Animal-derived fats: butter, lard coconut, peanut, sesame) Legumes (excluding lentils and beans): peas, peanuts Cooked lentils and beans, excluding soy beans or soy bean-derived products Non-cruciferous vegetables: artichokes, avocado, beets, carrots, celery, corn, Cruciferous (Brassicaceae) vegetables: broccoli, bok choy, cauliflower, cucumber, eggplant, garlic, ginger, fresh herbs, lettuce, mushrooms, parsnips, cabbage, kale, Brussels sprouts, bitter greens pears, potatoes, squash (acorn, butternut, spaghetti, summer, winter, spaghetti, yellow crooked neck, zucchini) Fresh fruit: bananas, apples, citrus, berries, melons, grapes, kiwi, mango, plums, Dried fruit pears, Beverages: mineral water, sulfited wines Beverages: fruit and vegetable juices coffee, tea, distilled spirits, beer Soy Products: soy milk, tofu, tempeh, miso, soy crisps, dried soy beans Snack foods: granola bars, popcorn, pretzels, potato chips, rice cakes Nuts and seeds: almonds, cashews, hazelnuts, pistachios, walnuts, nut Nuts and seeds: chestnuts, pecans butters, flax, hemp, pumpkin, sunflower, and sesame seeds 1High-sulfur food defined as SAA content > 0.5g/100g food item. Sulfur content derived from total methionine and cysteine (g) per 100 g of food or beverage, as specified by USDACondiments: National Nutrient mayonnaise Database for Standard Reference, Release 288. All dairy productsCondiments: included insalad High-S dressings; diet due ketchup, to milk fat mustard, content. canned sauces and gravies 2SAA concentration based on measurement after cooking. Mean Intake of Select Nutrients (NDSR Data) Nutrient Baseline High-S Diet Washout Low-S Diet Total Energy, kcal 1700 ± 454 1690 ± 600 1600 ± 593 1679 ± 367 Total Fat, g 73.6 ± 9.77 91.7 ± 39.1 64.1 ± 26.0 70.3 ± 23.3 % kcal from fat 39.7 ± 9.51 48.4 ± 8.44 35.4 ± 7.47 29.8 ± 9.65 Cholesterol, mg 270 ± 122 386 ± 240 238 ± 146 101 ± 73.4 Saturated fat, g 23.7 ± 5.22 28.2 ± 14.6 26.5 ± 13.0 27.6 ± 14.3 Monounsaturated fatty acids, g 22.7 ± 2.46 36.6 ± 17.1 19.7 ± 6.91 22.5 ± 5.73 Polyunsaturated fatty acids, g 21.1 ± 10.9 20.8 ± 12.9 12.8 ± 5.38 15.5 ± 2.91 Omega-3 fatty acids, g 3.6 ± 2.04 3.13 ± 1.59 1.29 ± 0.77 1.67 ± 0.31 Total Carbohydrate, g 185 ± 82.6 109 ± 59.1 206 ± 88.3 242 ± 41.0 % kcal from carbohydrate 42.8 ± 10.4 24.8 ± 6.83 49.9 ± 6.88 56.6 ± 7.26 Fiber, g 18.2 ± 1.57 11.5 ± 5.22 8.53 ± 0.94 20.6 ± 1.65 Total Protein, g 83.1 ± 24.0 a111 ± 25.4 54.1 ± 15.7 a33.7 ± 13.4 % kcal from protein 19.1 ± 1.15 26.8 ± 8.65 13.6 ± 2.55 7.05 ± 1.64 Animal protein, g 43.2 ± 8.71 94.3 ± 21.8 37.0 ± 9.79 3.90 ± 3.88 Vegetable protein, g 39.9 ± 30.9 17.0 ± 6.68 17.1 ± 4.10 29.8 ± 9.65 Methionine, g 1.75 ± 0.21 a2.69 ± 0.71 1.3 ± 0.40 a0.557 ± 0.250 Cystine, g 1.16 ± 0.34 a1.31 ± 0.270 0.768 ± 0.268 a0.571 ± 0.233 Results Results Methodology Optimization

• Simulation of anaerobic conditions in colon: ﬂushing of inert gas

• Incubation time for detectable gas concentrations

• Sample storage?

Future Directions

• Pending 16S rRNA sequencing data • Re-run interventions with longer time period • Measure H2, CH4, CO2 • Single study participant was not CH4 producer Acknowledgement

Thank you for all your help and support! • Michael Sadowsky • Alexandar Khoruts • Matthew Hamilton • Allison Dostal • Jean-Paul Schirle-Keller • Kurt Spokas • Martin du Saire

• Funding and opportunity provided by T35 Medical Student Summer Research • Thank you to Dr. Daniel Mueller and Andrea Stewart!