The ABCs of PPM

What does “gluten-free” really mean?

Emily Kaufman • Celiac Awareness Tour, Cleveland 2012

Let’s Talk About:

•  What does gluten-free mean according to different countries / organizations?

•  What’s the deal with oats? •  Why doesn’t gluten-free mean zero gluten?

Is this necessarily a bad thing? •  What’s a PPM, anyway? •  How can gluten be detected?

Gluten-free in the USA

•  No official federal guidelines – yet! •  Certification available from GFCO

(GIG), CSA, NFCA/QAI, etc. •  Differing requirements for oats •  Minimum thresholds of 5 or 10ppm

Gluten-Free Around the World

Codex Alimentarius Jointly organized commission of the World Health Organization & Food and Agriculture Organization of the United Nations. Set international food standards; their gluten regulations are observed in much of Europe, including all EU member countries:

–  < 20ppm = gluten-free –  20ppm < < 100ppm = very low gluten –  Oats to be decided by individual countries –  “Specially processed to remove gluten” WBRO OK –  Fermented/Hydrolyzed WBRO not specified

GF Around the World (cont.’) •  Argentina

–  < 10ppm = gluten-free / no oats

•  Brazil –  Labels must say either “gluten-free” or “contains

gluten” but there is no official ppm threshold

•  Australia/New Zealand –  < 3ppm = gluten-free (no detectable gluten) / no

oats –  Coeliac Australia certifies foods that are < 20ppm

•  Canada

–  < 20ppm = gluten-free / no oats –  Canadian Celiac Association considers oats safe

What’s the deal with oats? •  Avena is a genus of grass. Humans mostly eat avena sativa

and avena nuda. •  Subcategories: Jim, Rio Grande, INO9201 •  Cross-contamination in the fields / processing is the most

common reason oats are not gluten-free, HOWEVER: •  A 2007 study showed celiac T-cells responded differently to various strains of pure oats. •  A 2011 study matched positive results (G12 anti-

gliadin ELISA) from some pure oat samples with reactions in celiac T-cells.

•  SO: some strains of oat have some inherent toxicity, although even the most toxic avenin

is much less toxic than gliadin (wheat).

Why gluten-free ≠ 0 gluten

•  The amount of gluten we can detect has dropped drastically over the years.

•  It still isn’t at zero, but even if it were: – Cross-contamination is not homogenous – Studies indicate that minute amounts of

gluten are not harmful to celiac patients – The most sensitive, specific tests (ELISAs)

are not practical for day-to-day testing

What’s a PPM, anyway? PPM = Parts Per Million = mg per kg 20 mg/kg = 0.00032 oz. per lb. (appx.)

The most commonly-accepted study on gluten toxicity established a safe threshold at 10mg of gluten per day (Catassi et al 2007) How to translate PPM to daily diet? Depends on the diet! •  Half a pound of food at 20ppm ✔ •  A vitamin that contains 40ppm ✔ •  A bag of cookies at 10ppm ✔ •  8 slices of “typical” GF bread at 20ppm ✔

•  1/2000 of a slice of Wonder Bread ✔ ?

Comprises several different proteins, including the glutelin and the prolamin: Glutelins = Barley glutelin, etc.

Wheat is glutenin Prolamins = Wheat, gliadin

Barley, hordein Rye, secalin

Just within gliadin, we have multiples: alpha(α), omega(ω), gamma(γ) Genetic diversity makes for slight differences in the structure of some of these components from one plant to another (different alleles). Of all this protein, only a few tiny portions are responsible for most of the response in most people with celiac disease. Most common trigger: 33-mer peptide on the α-gliadin molecule

What exactly is gluten?

If Waldo were gluten… @1990: the Skerritt antibody, detects the ω-gliadin, heat-stable, underestimates barley @2003: the R5 detects certain small fragments of the α-gliadin, Codex type 1 (2006), overestimates barley, trouble with hydrolyzed/fermented grains @2008: G12, detects a fragment of the 33mer peptide (the pom-pom on Waldo’s hat)

There are others, but these are the most commonly seen. As scientists have learned more and more about gluten, various weaknesses within the antibodies have been corrected.

The α-gliadin molecule

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33-mer peptide

G12 binding

Antibody-Based Tests: LFD vs. ELISA

Both types of test incorporate •  an antibody •  a sample to test •  an extraction process to separate out

the gluten •  an extraction solution •  something that changes color

Lateral  Flow  Tests  The sample is applied to the end of a test strip. The test strip is essentially a piece of paper, with antibody and colored beads preloaded. The solution, full of maybe-gluten, flows laterally along the strip. If the antibody finds something to latch onto, the colored beads get activated: a positive test line appears.

A lateral flow test is qualitative: it can only tell you yes or no. LFDs are generally considered easiest and most cost-effective to produce, use, and analyze.

ELISA (Enzyme-Linked ImmunoSorbent Assay) Quantitative: results are a specific ppm. Antibody often sits at the bottom of a PVC tray (instead of on paper). More involved extraction and application processes: at strategic times and temperatures, various solutions are applied to the tray and washed off. If the antibody finds what it’s looking for, a linked enzyme changes the color of the enzyme’s substrate, indicating the presence of gluten. Not read by the naked eye: a machine compares the intensity of the color of each sample to those of positive and negative controls. Sandwich, Competitive ELISAs have different constructions and strengths

Testing Methods in Sum •  Both ELISA and Lateral Flow tests play important roles in

preventing cross-contamination of gluten-free foods. •  Lateral Flow tests are generally quicker and less

expensive than ELISAs, but provide less information and have fewer variables to tinker with.

•  There are different types of ELISA; a reputable lab will recommend one or the other (or multiple!) depending on the sample.

•  ELISA and Lateral Flow are simply categories. Each can be created with different antibodies, extraction solutions, etc. So, knowing that a food was ELISA-tested does not tell us much about whether or not it is safe to eat.

•  Mass Spectrometry & Polymerase Chain Reaction (PCR) methods are being developed for lab use/

Thanks for listening!

For more info •  @GlutenTox •  facebook.com/GlutenTox •  glutentox.com