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

Making Sense of Science

Megan Tichy, Ph.D.Lecturer, Texas A&M University

Toxic grains Scientific and non-scientific names

Quantification Parts per million

Gluten Chemical structure

Processing Distillation

Highlights

• Classification based on physical, behavioral or genetic diversity• Subject to revisions

Scientific names

Human Pea Wheat

Domain Eukarya Eukarya Eukarya

Kingdom Animalia Plantae Plantae

Phylum Chordata Magnoliophyta Magnoliophyta

Class Mammalia Magnoliopsida Lilliopsida

Order Primates Fabales Cyperales

Family Hominidae Fabaceae Poaceae

Genus Homo Pisum Triticum

Species H. sapiens P. sativum T. aestivum

More

sp

ecifi

c

Mnemonic device: Kids Prefer Cheese Over Fried Green Spinach

Poaceae

Festucoideae

Oryzeae - rice

Triticeae - wheat, rye, barley

Aveneae - oats

Panicoideae Andropogoneae - sorghum

Tripsaceae - corn

Paniceae - millet

KINGDOM – Plantae – plantsPHYLUM – Magnoliophyta – flowering plantsCLASS – Liliopsida – monocotyledonsORDER – Cyperales – grasses/sedges

The grass family

FAMILY

SUBFAMILY

TRIBE

Donald D. Kasarda, Ph.D., Research Chemist (Retired), USDA http://wheat.pw.usda.gov/ggpages/index.shtml

WheatTriticum aestivum

BarleyHordeum vulgare

RyeSecale cereale

Images: Home-Grown Cereals Authority (HGCA); http://www.hgca.com

Toxic grains

Durum

Hard Red Winter

Hard Red Spring

Hard White Wheat

Soft Red Winter

Soft White Wheat

Modern wheat varieties

Atta whole wheat, stone-grinded Bulgur whole wheat, precooked, dried, cracked, and sifted Couscous moistened semolina, rolled and shaped,

coated with finely ground wheat four Farina endosperm milled to fine granular consistency, sifted Seitan washed dough; water removes starch Semolina inner endosperm of wheat not ground into flour

Images: Wikipedia commons

Seitan (Taiwan)

Wheat products

Atta (India) Farina

Ancient forms of wheat

Einkorn Triticum monococcum DiploidEmmer (farro) Triticum dicoccum TetraploidSpelt (dinkel) Triticum spelta Hexaploid

Kamut Triticum turanicum Tetraploid

Genetics: Diploid = 14 chromosomesTetraploid = 28 chromosomesHexaploid = 42 chromosomes

More chromosomes = easier to breed varieties suitable for non-ideal soils/climates

Wheat proteins

Protein Soluble in

Albumin WATER - neutral pH

Globulin WATER - salt solutions

Prolamin ETHANOL - 70-90% alcohol

Glutelin NONE OF THE ABOVEdilute acids/bases, detergents, or reducing agents

Gluten

Cause of most wheat allergies

“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.

Gluten proteins

GLUTEN

GLIADINS

GLUTENINS

HMW LMW

All coded by different genes (DNA)

Prolamine = proline + glutamine

Grain Prolamin fraction % total protein

Wheat Gliadin 69

Rye Secalin 30-50

Barley Hordein 46-52

Oats Avenin 16

Millet Panicin 40

Corn Zien 55

Rice Orzenin 5

Sorghum Kafirin 52

Grain proteins

toxic safe still questionedcontaminated? pure?

BranRich source of magnesium and iron. ¼ cup contains 6 grams fiber.

GermEmbryo

vitamin E, magnesium, riboflavin, thiamin,

phosphorus, niacin, iron and zinc. Some fat and protein.

Manufacturers remove the germ and bran to make refined bread flour

Anatomy of a kernel

EndospermStorage tissuestarch, gluten

Whole-grain: endosperm, bran, and embryo (all three parts of the kernel!) left intact

flour

dough

starch (sugars)75%

gluten (protein)12% (varies)

Wheat flour: gluten and starch

H2O

C6H12O6 + 6 O2 → 6 CO2 + 6 H2O + energy

glucose + oxygen → carbon dioxide + water

doughyeast bread

Image: http://www.exploratorium.edu/cooking/bread/activity-gluten.html

• 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 g gluten 1/8 tsp 33 mg gluten

How much gluten?

Measuring spoon image: http://www.tubularspices.com/accessories/spoons.asp

How much is too much?

Codex Alimentarius (WHO/FAO), has two limits for “gluten-free”: 0.02% gluten content - rendered gluten-free0.002% gluten content - naturally gluten-free food

Normal gluten intake by healthy individuals = 13 g gluten per dayCompare – 50 mg (0.05 g) vs. 13 gNormal intake is 260 times as much as the suggested threshold

“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*

A person could ingest 3-6 mg gluten per day by consuming 6-8 ounces of “naturally gluten free” grain food

*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

0.002% = 20 parts per million or ppm

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

per cent means one part per hundred X 100 =

A part per hundred (%)

One bushel of wheat (60 lbs) contains 1 million individual kernels

A part per million (ppm)

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:A) 4 days

B) 10 weeks C) 2 years

20 minutes

4 days

1 hour

60 minutes

1 day

24 hours20

5760

= 0.003472= 3,472 ppm

=

20 minutes

2 years

1 hour

60 minutes

1 day

24 hours20

10512001 year

365 days

= 0.00001902= 19 ppm

=

Quiz answer

20 ppm

20 seconds in 11.5 days

20 inches in 15.8 miles

20 ounces in 62,500 pounds

20¢ in $10,000

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

Using ppm to calculate quantity gluten

ppm gluten

2 g

Tsp non-dairy GF creamer

20 g

10 GF crackers

200 g

4 slices GF bread

20 0.04 mg 0.4 mg 4 mg

50 0.1 mg 1 mg 10 mg

100 0.2 mg 2 mg 20 mg

200 0.4 mg 4 mg 40 mg

Reminder: The ingestion of contaminating gluten should be kept lower than 50 mg/day in the treatment of CD

Proteins

Contain carbon, oxygen, hydrogen, nitrogen, often sulfur

Broken down to 22 naturally occurring amino acids

Starch vs. Protein

Starches

Contain carbon, oxygen, hydrogen

Broken down to only one material, glucose

HN

NH

HN

O

O

O

NH

HN

HN

HO

O

O

O

NH

O

OH SH

OH

SH

O

HOOH

OHO

HOHO

OOH

OH

O

HOOH

OHO

OHO

OOH

OH

O

glucose

aminoacid

I Have Received Much Kudos For Learning These Very Well

Mnemonic device:

O

OHH2N

IsoleucineIleI

HistidineHisH

OHO

NH2

HN

N

ArginineArgR

O

OHH2N

HN

HN

NH2 MethionineMetM

O

OHH2N

S

LysineLysK

O

OHH2NH2N

PhenylalaninePheF

O

OHH2N

LeucineLeuL

O

OHH2N

ThreonineThrT

O

OHH2N

OH

ValineValV

O

OHH2N

TryptophanTrpW

O

OHH2N

NH

Essential amino acids (from diet)

All amino acids have a 1-letter abbreviation

Proteins are polymers of amino acids with directionalityC (carboxy)-terminus → N (amino)-terminus

“Beads on a string”

Protein primary structure

Stability: Proteins vary widely in their stability to heatProline is known to provide stability to proteins

MKTFLILALLAIVATTATTAVRVPVPQLQPQNPSQQQPQ

EQVPLVQQQQFLGQQQPFPPQQPYPQPQPFPSQQPYLQL

QPFLQPQLPYSQPQPFRPQQPYPQPQPQYSQPQQPISQQQ

QQQQQQQQQQQQQQQQIIQQILQQQLIPCMDVVLQQHN

IVHGKSQVLQQSTYQLLQELCCQHLWQIPEQSQCQAIHN

VVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRP

SQQNPQAQGSVQPQQLPQFEEIRNLARK

Gliadin primary structure (sequence)

Q: Why does gliadin survive in the presence of enzymes and heat?

MKTFLILALLAIVATTATTAVRVPVPQLQPQNPSQQQPQ

EQVPLVQQQQFLGQQQPFPPQQPYPQPQPFPSQQPYLQL

QPFLQPQLPYSQPQPFRPQQPYPQPQPQYSQPQQPISQQQ

QQQQQQQQQQQQQQQQIIQQILQQQLIPCMDVVLQQHN

IVHGKSQVLQQSTYQLLQELCCQHLWQIPEQSQCQAIHN

VVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRP

SQQNPQAQGSVQPQQLPQFEEIRNLARK

Reference: L. Shan, O. Molberg and I. Parrot et al., Structural basis for gluten intolerance in celiac sprue, Science 297 (2002), pp. 2275–2279.

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.

Protein folding

amino acids

binding site

Protein function requires folding

•Structure: keratin •Transport: hemoglobin•Storage: myoglobin•Hormones: insulin •Catalysis: lactase

5 common protein functions:

Just a few “beads” from the string ≠ gluten

The average person consumes• 10 - 20 g bound glutamate/day• 0.5 - 1.5 g MSG/day

MSG ≠ gluten

Na+

NH2

O

-O

O

OH

monosodium glutamate(both L and D)

NH2

O

H2N

O

OH

L-glutamine

NH2

O

HO

O

OH

L-glutamic acid

L versus D• In higher mammals, all proteins contain “L” amino acids. • During hydrolysis “L” → “D” • The “D” form is tasteless

Other small molecules ≠ gluten

O

N

N

NN

O The structure of caffeine

Not all symptoms are related to celiac disease

Calcium oxalate can form crystals which eventually grow into kidney stones

Oxalate binds with calcium in your urinary tract

Ca2+OO-

O-

O

The structure of calcium oxalate

200-300 milligrams daily appears to be safe

Starbucks’ Tall Coffee = 260 milligrams

How do we test for gluten?ELISA – Enzyme Linked Immunosorbent AssayELISA detection limit between 1-2 ppm, with <0.5 ppm reported*

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

Hydrolyzed wheat starch

*Sousa et al. Am J Clin Nut, Vol. 87, No. 2, 405-414, February 2008

North America – starch products usually derived from corn

Hydrolysis = hydro (water) + lysis (cleavage/breakage) A chemical reaction in water that breaks down wheat starch

Alcoholic fermentation

A vessel allows carbon dioxide to escape, but prevents outside air from coming in

C6H12O6 → 2 C2H5OH + 2 CO2

glucose → ethanol + carbon dioxide

Without oxygen present:

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

Safe liquors

Distillation: separation based on volatility

Volatile

Non-volatile

Volatility - how readily substances become gasesStronger intermolecular forces decrease volatilityIt takes more energy to break these molecules apart

NaCl

H3N CH C

CH2

O

O

CH2

C

NH2

O

glutamine

H2C

CH3HO

ethanol

CCH3

O

O

H2C

H3C

ethyl acetateH3C

C

O

CH3

acetone

H2O water

Also not volatile:large peptides (proteins)starches, any polymer

Not solely based on sizeof the molecule

Ethyl acetateMolecular Weight: 88 g/moleBoiling Point: 77 C C

CH3

O

O

H2C

H3C

H3C

C

O

CH3

EthanolMolecular Weight: 32.04 g/moleBoiling Point: 64.7 C

AcetoneMolecular Weight: 58.08 g/mole Boiling Point: 56.5 C

ButanolMolecular Weight: 74.12 g/moleBoiling point: 117.7 C

H2C

CH3HO

H2C

OHCH2

H2C

H3C

Incr

easi

ng b

oili

ng p

oin

tVolatility ≠ size

3 C’s1 O

2 C’s1 O

4 C’s1 O

4 C’s1 O

Simple distillation procedure

The separated liquid (distillate) drips out into a new chamber

Volatile portion enters gas phase Tubes are cooled

gas → liquid

Ethanol gas floats upcirculates throughcondensing tubes

Liquid heated in boiling chamber

Peptides/amino acidsdo not evaporate

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

THANK YOU!

Brazos Valley Gluten Intolerance GroupDenise Fries, Taylor Jensen Julie Hoyle (secretary), JenSara Boswell Kim Melissari (treasurer), LaurenMonica Michalka

FamilyShane (husband), Nathan (son)Karen Jones & FamilyMcLean family – Rochester, NYSantiago family – Bryan, TX

GIG mentorsCynthia Kupper, RDBetty Barfield

Texas A&M UniversityDr. Ganesa Gopalakrishnan