Making Sense of Science Megan Tichy, Ph.D. Lecturer, Texas A&M University

1. Making Sense of ScienceMegan Tichy, Ph.D.Lecturer, Texas A&M University

2. Highlights • Toxic grains • Scientific and non-scientific names • Quantification • Parts per million • Gluten • Chemical structure • Processing • Distillation

3. Scientific names • Classification based on physical, behavioral or genetic diversity • Subject to revisions More specific Mnemonic device: Kids Prefer Cheese Over Fried Green Spinach

4. The grass family KINGDOM – Plantae – plants PHYLUM – Magnoliophyta – flowering plants CLASS – Liliopsida – monocotyledons ORDER – Cyperales – grasses/sedges TRIBE Oryzeae - rice SUBFAMILY Festucoideae Aveneae - oats FAMILY Triticeae - wheat, rye, barley Poaceae Paniceae - millet Andropogoneae - sorghum Panicoideae Tripsaceae - corn Donald D. Kasarda, Ph.D., Research Chemist (Retired), USDA http://wheat.pw.usda.gov/ggpages/index.shtml

5. Toxic grains Modern wheat varieties Wheat Triticum aestivum Durum Barley Hordeum vulgare Hard Red Winter Hard Red Spring Hard White Wheat Rye Secale cereale Soft Red Winter Soft White Wheat Images: Home-Grown Cereals Authority (HGCA); http://www.hgca.com

6. Wheat products Atta whole wheat, stone-grinded Bulgur whole wheat, precooked, dried, cracked, and sifted Couscousmoistened semolina, rolled and shaped, coated with finely ground wheat four Farina endosperm milled to fine granular consistency, sifted Seitanwashed dough; water removes starchSemolina inner endosperm of wheat not ground into flour Atta (India) Farina Seitan (Taiwan) Images: Wikipedia commons

7. Ancient forms of wheat Genetics: Diploid = 14 chromosomes Tetraploid = 28 chromosomes Hexaploid = 42 chromosomes More chromosomes = easier to breed varieties suitable for non-ideal soils/climates Einkorn TriticummonococcumDiploid Emmer (farro) Triticum dicoccum Tetraploid Spelt (dinkel) Triticum spelta Hexaploid Kamut Triticum turanicum Tetraploid

8. Wheat proteins Cause of most wheat allergies Gluten

9. Gluten proteins Prolamine = proline + glutamine All coded by different genes (DNA)     HMW LMW “It is highly probable that glutenin proteins are toxic. Attempts to breed wheat with satisfactory baking properties tolerated by coeliac patients will be very difficult.” Eur J Gastroenterol Hepatol. 2006 May;18(5):483-91.

10. Grain proteins toxic safe still questioned contaminated? pure?

11. Anatomy of a kernel Manufacturers remove the germ and bran to make refined bread flour Endosperm Storage tissue starch, gluten Bran Rich source of magnesium and iron. ¼ cup contains 6 grams fiber. Germ Embryo vitamin E, magnesium, riboflavin, thiamin, phosphorus, niacin, iron and zinc. Some fat and protein. Whole-grain: endosperm, bran, and embryo (all three parts of the kernel!) left intact

12. Wheat flour: gluten and starch flour H2O dough starch (sugars) 75% gluten (protein) 12% (varies) dough yeast bread C6H12O6 + 6 O2 → 6 CO2 + 6 H2O + energy glucose + oxygen → carbon dioxide + water Image: http://www.exploratorium.edu/cooking/bread/activity-gluten.html

13. How much gluten? • Flour contains 10-15% protein • The protein is 80% gluten • Q: If 1 slice of bread uses 40 g flour • how much gluten is in it? • A: solve for protein 40 g X 0.13 = 5.2 g protein • solve for gluten 5.2 g X 0.8 = 4 g gluten • Q: How many mg gluten is in 1/8 teaspoon flour? • A: Conversion for wheat flour 1 tsp = 2.5 g • 1/8 tsp all purpose flour = 0.31 g flour • 0.31 g X 0.13 X 0.8 = 0.033 ggluten • 1/8 tsp  33 mg gluten Measuring spoon image: http://www.tubularspices.com/accessories/spoons.asp

14. How much is too much? “Ingestion of contaminating gluten should be kept lower than 50 mg/day in the treatment of CD” – C. Catassi et al. AmJ Clin Nutr 2007;85:160–6. Some people are far more sensitive* Normal gluten intake by healthy individuals = 13 g gluten per day Compare – 50 mg (0.05 g) vs.13 g Normal intake is 260 times as much as the suggested threshold Codex Alimentarius (WHO/FAO), has two limits for “gluten-free”: 0.02% gluten content - rendered gluten-free 0.002% gluten content - naturally gluten-free food A person could ingest 3-6 mg gluten per day by consuming 6-8 ounces of “naturally gluten free” grain food 0.002% = 20 parts per million or ppm *Reference: A milligram gluten a day keeps the mucosal recovery away: a case report, F. Biagi, J. Campanella and S. Martucci et al., Nutr Rev 62 (2004), pp. 360–363

15. A part per what? • Examples of a part per twelve • One egg out of a dozen • One cupcake out of a dozen • One pane from this stained-glass window A part per hundred (%) per cent means one part per hundred X 100 = A part per million (ppm) One bushel of wheat (60 lbs) contains 1 million individual kernels

16. A part per million (ppm) • Use % for larger fractions • 1 part per 100 = 0.01 = 1% • Use ppm for smaller fractions • 1 part per 1,000,000 = 0.000001 = 0.0001% • Calculating ppm: • Start with the fraction (part/whole) • Rather than 2 decimal places to the right → % • Move 6 decimal places to the right → ppm • 20 ppm = 20 minutes in: • 4 days • B) 10 weeks • C) 2 years

17. Quiz answer 20 minutes 1 hour 1 day 20 = 60 minutes 4 days 24 hours 5760 = 0.003472 = 3,472 ppm 20 minutes 1 hour 1 day 1 year 20 = 1051200 365 days 60 minutes 2 years 24 hours = 0.00001902 = 19 ppm

18. Perspective: How much is 20 ppm? Tiny amounts are biologically significant! Most fish need  4 ppm dissolved oxygen to survive; Bass and trout seek out waters with 9-12 ppm oxygen

19. Using ppm to calculate quantity gluten Reminder: The ingestion of contaminating gluten should be kept lower than 50 mg/day in the treatment of CD

20. Starch vs. Protein Starches Contain carbon, oxygen, hydrogen Broken down to only one material, glucose Proteins Contain carbon, oxygen, hydrogen, nitrogen, often sulfur Broken down to 22 naturally occurring amino acids glucose amino acid

21. Essential amino acids (from diet) All amino acids have a 1-letter abbreviation Mnemonic device: IHave Received Much Kudos For Learning These Very Well

22. “Beads on a string” Protein primary structure Proteins are polymers of amino acids with directionality C (carboxy)-terminus → N (amino)-terminus Stability: Proteins vary widely in their stability to heat Proline is known to provide stability to proteins

23. Gliadin primary structure (sequence) MKTFLILALLAIVATTATTAVRVPVPQLQPQNPSQQQPQ EQVPLVQQQQFLGQQQPFPPQQPYPQPQPFPSQQPYLQL QPFLQPQLPYSQPQPFRPQQPYPQPQPQYSQPQQPISQQQ QQQQQQQQQQQQQQQQIIQQILQQQLIPCMDVVLQQHN IVHGKSQVLQQSTYQLLQELCCQHLWQIPEQSQCQAIHN VVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRP SQQNPQAQGSVQPQQLPQFEEIRNLARK MKTFLILALLAIVATTATTAVRVPVPQLQPQNPSQQQPQ EQVPLVQQQQFLGQQQPFPPQQPYPQPQPFPSQQPYLQL QPFLQPQLPYSQPQPFRPQQPYPQPQPQYSQPQQPISQQQ QQQQQQQQQQQQQQQQIIQQILQQQLIPCMDVVLQQHN IVHGKSQVLQQSTYQLLQELCCQHLWQIPEQSQCQAIHN VVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRP SQQNPQAQGSVQPQQLPQFEEIRNLARK Q: Why does gliadin survive in the presence of enzymes and heat? Reference: L. Shan, O. Molberg and I. Parrot et al., Structural basis for gluten intolerance in celiac sprue, Science297 (2002), pp. 2275–2279.

24. Protein folding Secondary structure: Regularly repeating local structures stabilized by hydrogen bonds. Ex: alpha helix, beta sheet Tertiary structure: Overall shape or “fold”; the spatial relationship of the secondary structures relative to one another Quaternary structure: Results from interaction of more than one protein molecule (subunit) which functions as part of the larger assembly or protein complex.

25. amino acids binding site Protein function requires folding 5 common protein functions: • Structure: keratin • Transport: hemoglobin • Storage: myoglobin • Hormones: insulin • Catalysis: lactase Just a few “beads” from the string ≠ gluten

26. MSG ≠ gluten • The average person consumes • 10 - 20 g bound glutamate/day • 0.5 - 1.5 g MSG/day • L versus D • In higher mammals, all proteins • contain “L” amino acids. • During hydrolysis “L” → “D” • The “D” form is tasteless

27. Other small molecules ≠ gluten Not all symptoms are related to celiac disease The structure of caffeine 200-300 milligrams daily appears to be safe Starbucks’ Tall Coffee = 260 milligrams The structure ofcalciumoxalate Oxalate binds with calcium in your urinary tract Calcium oxalate can form crystals which eventually grow into kidney stones

28. Hydrolyzed wheat starch Hydrolysis = hydro (water) + lysis (cleavage/breakage) A chemical reaction in water that breaks down wheat starch North America – starch products usually derived from corn • Any of the following can be made from wheat starch: • Maltodextrin, glucose syrup, dextrose • Poly-ols, sugar alcohols (sorbitol, xylitol, mannitol etc…) • Lactic acid, citric acid, acetic acid (vinegar), ascorbic acid How do we test for gluten? ELISA – Enzyme Linked Immunosorbent Assay ELISA detection limit between 1-2 ppm, with <0.5 ppm reported* *Sousa et al. Am J Clin Nut, Vol. 87, No. 2, 405-414, February 2008

29. Alcoholic fermentation Safe liquors Wine/brandy - natural sugars present in fruits Rum - cane sugar or molasses Whiskey - amylase-treated grain Vodka - amylase-treated grain or potatoes Gin - grain infused with juniper berry A vessel allows carbon dioxide to escape, but prevents outside air from coming in Without oxygen present: C6H12O6 →2 C2H5OH+ 2 CO2 glucose →ethanol+ carbon dioxide

30. Distillation: separation based on volatility Volatility - how readily substances become gases Stronger intermolecular forces decrease volatility It takes more energy to break these molecules apart Volatile Non-volatile Also not volatile: large peptides (proteins) starches, any polymer Not solely based on size of the molecule

31. Volatility ≠ size Acetone Molecular Weight: 58.08 g/mole Boiling Point: 56.5 C 3 C’s 1 O 2 C’s 1 O Ethanol Molecular Weight: 32.04 g/mole Boiling Point: 64.7 C Increasing boiling point Ethyl acetate Molecular Weight: 88 g/mole Boiling Point: 77 C 4 C’s 1 O Butanol Molecular Weight:74.12 g/mole Boiling point: 117.7 C 4 C’s 1 O

32. Simple distillation procedure Ethanol gas floats up circulates through condensing tubes Volatile portion enters gas phase Tubes are cooled gas → liquid Liquid heated in boiling chamber Peptides/amino acids do not evaporate The separated liquid (distillate) drips out into a new chamber

33. Conclusions • There are MANY other names for wheat • Spelt is NOT an alternative for wheat! • A ppm is not a quantity • 20 ppm can be many different quantities, depends on how much you ate! • Gluten is a highly stable protein fraction • Several different fragments of gliadin and glutenin are toxic • Small molecules do not mimic gluten, but can have negative • health effects of their own • Distillation is separation based on volatility • Distilled products are gluten-free as long as gluten was not added post-distillation

34. THANK YOU! Brazos Valley Gluten Intolerance Group Denise Fries, Taylor Jensen Julie Hoyle (secretary), Jen Sara Boswell Kim Melissari (treasurer), Lauren Monica Michalka GIG mentors Cynthia Kupper, RD Betty Barfield Texas A&M University Dr. Ganesa Gopalakrishnan Family Shane (husband), Nathan (son) Karen Jones & Family McLean family – Rochester, NY Santiago family – Bryan, TX