Wednesday, October 5, 2011

The Trouble With Measuring AGEs -- Butter and More

by Chris Masterjohn
This post is basically a technical footnote to my next post on advanced glycation endproducts (AGEs) and all subsequent posts on AGEs explaining why I will give preference to certain studies that use what I consider reliable methods for measuring these compounds.

In my previous posts, "Is Butter High in AGEs?" and "Is the Receptor for AGEs (RAGE) Really a Receptor for AGEs?" I was highly critical of immunological techniques for measuring AGEs.  These are methods where investigators will incubate a sample with antibodies that are supposedly specific to AGEs and then see how much antibody sticks.  It's kind of like measuring the length and width of a wall by throwing gum at it and seeing how much sticks. It's crude, but it works pretty well as long as each piece of gum sticks to each piece of the wall with roughly the same affinity.  The problem with measuring AGEs this way is that we have a wall with lots of different intricate topical features with different affinities for gum, and cheap gum that'll stick to anything.

The main alternative to immunological techniques is chromatography.  Chromatographic methods separate each individual compound in a sample, allowing the investigator to identify each compound and precisely measure its mass.  Thus immunological methods measure things in vague and arbitrary terms like "AGE units" (i.e., pieces of gum you threw at the wall) and chromatographic methods measure things in concrete and familiar terms like "milligrams."

You can read more about my specific objections in the blog posts I just linked to, but here I'd just like to take a brief moment to point out that I am hardly alone in my criticisms.
In my skepticism of immunological AGE measurements I am joined by such luminaries as PJ Thornalley, who has devoted his life's work to studying AGEs and their precursors.  Thornalley and his colleague Naila Rabbani have organized the only two symposia dedicated to the glyoxalase system, our main enzymatic defense against AGEs, over the past twenty years (December 1992 and July 2003) (1).  Together they published a review on measuring AGEs (2), in which they identified numerous problems with immunological techniques including questionable specificity of the antibodies and different affinities between the antibody and the AGE depending on what the AGE is attached to.

I was pleased to find earlier today that German AGE researcher Thomas Henle, in the chapter he wrote for the textbook Process-Induced Food Toxicants: Occurrence, Formation, Mitigation, and Health Risks (3), took the butter-is-high-in-AGEs researchers to task for exactly the same reason I did last October, and made similar criticisms of the most commonly cited study purporting to quantify just how much AGEs we absorb from our food (4):
It may be obvious in many cases that data reported for glycation compounds do not fulfill the basic requirements for a credible analysis even if published in "high-impact" journals.  It seems to be important to address this issue mainly because of recent publications reporting astonishing data of glycation compounds in foods.
The reference he gives for "astonishing data" is the 2004 version of the butter-is-high-in-AGEs database (5).  Referencing the same study later in the chapter, he continues:
Data for CML [a specific AGE] published in literature based on the sole use of an ELISA [a specific immunological technique], therefore, must be interpreted with care.  Unfortunately, corresponding reports gained much interest in context with nutritional consequences resulting from dietary CML, although many of the published data are somewhat dubious and therefore shall not be discussed further.1
That footnote leads to the following statement at the bottom of the page:
For instance, butter and olive oil, respectively, are reported to contain several hundredfold higher "amount" of CML when compared with the crust of whole wheat bread (265,000 IU/g or 120,000 U/g versus 730 U), but such high amounts of an amino acid in the lipid samples are virtually impossible.  Even if butter would contain a "protein," composed solely of CML, such high concentrations of CML would not be realistic.
Notice how he uses "amount" in quotation marks.  He ain't feelin' it either.  Regarding the most popular study cited purporting to quantify how much AGE we absorb from food, he has the following to say:
As mentioned earlier, this "pioneering" study is questionable from an analytical point of view, as an immunological assay based on an antibody against CML was used for quantification, and no information about the reliability of the assay in quantifying CML in plasma, urine, and foods is given.
Referring to both of these questionable studies, he continues:
When reviewing reports presenting evidence for detrimental effect of dietary AGEs, one cannot overlook that most of the studies report biological phenomena without any chemical characterization of the stimulating agent.  "AGEs" are generally taken as "anonymous" mixture of compounds, for which no reliable data are given.  Most of the studies refer to the aforementioned database when creating diets high or low in AGEs.  In other words, up to now, there is no study showing unambiguously any toxicological effect of one single glycation compound based on accepted structure-function assays.  Actual studies based on chemically characterized compounds are rare, however, in general report either on low bioavailability of glycation compounds or on quick elimination without accumulation in vivo.

Finally, he concludes:
Animal feeding studies or human intervention studies, in which effects of diets "high" or "low" in AGEs are presented, should only be accepted if the amount of individual glycation compounds in the said food items is quantified using reliable chromatographic techniques, ideally calibrated with pure reference standards.  "Calculations" of an AGE content on the basis of flawed data collections are out of the question.
There he goes with the quotation marks again.  Boy, it seems like someone's feathers got ruffled!  Suffice it to say, I am hardly the only one criticizing these immunological techniques and for the reasons given in this section, I will in all future posts on AGEs give preference to studies using appropriate chromatographic techniques to acquire reliable data.

Read more about the author, Chris Masterjohn, PhD, here.


1.  Rabbani N, Thornalley PJ.  The glyoxalase system -- From microbial metabolism, through ageing to human disease and multidrug resistance.  Semin Cell Dev Biol. 2011;22(3):261.

2.  Rabbani N, Thornalley PJ.  Quantitation of Markers of Protein Damage by Glycation, Oxidation, and Nitration in Peritoneal Dialysis.  Perit Dial Int. 2009;(29 Suppl 2):S51-6.

3. Henle T. Maillard Reaction of Proteins and Advanced Glycation End Products (AGEs) in Food.  In Stadler RH, Lineback DR, eds. Process-Induced Food Toxicants: Occurrence, Formation, Mitigation, and Health Risks.  Wiley Online Library, Published Online July 11, 2008.

4.  Goldberg T, Cai W, Peppa M, Dardaine V, Baliga BS, Uribarri J, Vlassara H.  Advanced glycoxidation end products in commonly consumed foods.  J Am Diet Assoc. 2004;104(8):1287-91

5.  Koschinsky T, He CJ, Mitsuhashi T, Bucala R, Liu C, Buenting C, Heitmann K, Vlassara H.  Orally absorbed reactive glycation products (glycotoxins): an environmental risk factor in diabetic nephropathy.  Proc Natl Acad Sci USA. 1997;94(12):6474-9.


  1. Interesting to say the least. Clinical testing that may reveal AGE's is variable in humans. Butter is not one I have seen to cause a massive shift in clinical markers

  2. One little problem with AGE.
    If you eat a product containing AGE, what happens with the AGE in the intestines? If it not is degraded into amino acids and sugar separately the AGE is probably not taken up by our intestines and thus excreted in faeces.
    If AGE by some mystery pathway enters the body how can the tRNA couple a false amino acid-glucose/fructose complex in the ribosomes to produce an AGE-protein?
    The only way you can get AGE in or on cells is by a nonezymatic glycosylation of an already existing protein in or on the cell.
    AGEing is due to the exreme excess of toxic carbogydrates in our food ordered by the authorities. According to the Swedish Food Authority I'm supposed to eat 480 g sugars, mainly glucose per day. I'm supposed to eat about 100 times more sugar than I have in my total blood volume of 5.6 L. If my blood has more than 30-50 mmol/L (=30-50 grams) glucose I'll die in acute glucose poisoning. So I'm supposed to eat at tenfold lethal amount of sugar each and every day. Thanks to my Creator I have the disaster preventing insulin in adequate amounts to save me from the "advise" of the authorities!
    It's interesting that the authorities seem to totally lack Physiology 101 as well as Biochemistry 101 and hormonologt 101 and also seems devoid of any common logic as well.
    If there are any AGE in your body (HbA1c) above 50 mmole/mole (5 % in elder litterature) then you have too much carbohydrates (a.k.a. suger, glucose, fuctose) in your food.
    We all know that protein and fat are essential, delivering vitamins and minerals as well. We all know that carbohydrates are nonessential, "empty calories" lacking micronutrients compared to food of animal origin.
    So why dilute real food with "empty calories"?

  3. Thanks John.

    Bjorn, I just wrote an extensive post on this:

    Some AGEs are absorbed and some are not. Those that are absorbed seem to be quickly urinated out. There is no evidence that dietary AGEs can be incorporated into proteins and I am not aware of any AGE researchers who assert that this is possible.

    AGEs are not formed from "glycolsylation" of proteins. "Glycosylation" is a term reserved for the enzymatic addition of sugars to proteins which is an absolutely essential process that we cannot live without.

    AGEs are also not formed, for the most part, by direct "glycation" of proteins, which is the term you are looking for. Most AGEs are formed from a reaction between dicarbonyls and proteins, and yes, glycation by glucose plays a role in producing dicarbonyls, but it is hardly the only factor.

    There is no evidence to my knowledge that endogenous AGE formation is promoted by excessive consumption of carbohydrates. Please see the post I linked to above.

    Carbohydrates are physiologically essential. They are not dietarily essential. But that doesn't say anything about what's optimal.



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