Advances in Advanced Glycation Endproducts (AGEs)

GODIC, MODIC, DOGDIC, and Glarg. GOLD, MOLD, DOLD, and CROSSPY. ALI, vesperlysine, FFI and glucosepane. These are the nicknames for just a few members of a class of molecules called advanced glycation endproducts (AGEs).

AGEs, the structures of which are a beautiful combination of stereochemistry, asymmetry, and complexity, are a growing subject of study in medicine. Research has linked these molecules to a number of diseases, including diabetes mellitus, nephropathy, neuropathy, renal insufficiency, athersclerosis, and Alzheimer’s disease. This suggests that they may underlie the etiology of a wide array of diseases.

But is that the whole story – that AGEs are poisons? Before we delve any further, we must first explore what exactly these molecules are.

What are AGEs?

AGEs are proteins or lipids that have been modified in the presence of sugars, without the help of enzymes. In chemistry, this reaction with sugars is classified as glycation.

The first step in the natural synthesis of a protein-based AGE usually happens through what is called a Maillard (pronounced my-yard) reaction, which involves heating amino acids – the building blocks of proteins – along with saccharides – the building blocks of sugars. This type of reaction occurs during cooking, such as when you caramelize sugar and milk, when you malt barley into whiskey, and even when soft white dough is baked into the familiar brown of pretzels. In fact, researchers have successfully collected AGEs from pretzels.

When the Maillard reaction joins the sugar and the amino acid, the product is called a Schiff base. This base is not very stable and readily moves its bonds and hydrogens in the Amadori rearrangement. After the rearrangement, each AGE has its own distinct set of transformations to convert, irreversibly, the Amadori product into the final, stable AGE structure. We currently know of several dozen AGEs that occur in the body, but the total number of AGEs is believed to be tenfold higher.

Incredibly, all of this happens without the help of enzymes, nature’s catalysts. When sugars and proteins are brought together in the cell, the reactions occur spontaneously. However, the reactions are often quite slow. When experimenters performed in vitro reactions to investigate AGE formation in simulated normal intracellular conditions of 37ºC and pH 7.4, it took several months to observe successful production of the molecules of interest. Yet despite this slow speed, the fact remains that whenever proteins and sugars come into contact in your body, AGEs form.

AGEs in Disease

Though there remain many unanswered questions about AGEs, one thing we do know is that their formation becomes increasingly prevalent with age of the constituent parts. Because AGE formation takes so long, short-lived proteins are unlikely to become glycated. Proteins that have longer lifespans, however, like the collagen of your skin or the myelin of neurons, are not so fortunate. These proteins naturally interact with more sugars for longer periods, making them much more likely to undergo the glycation process, often multiple times.

Unsurprisingly then, many health effects associated with AGEs involve aging proteins. Some studies suggest that it is even possible to see the effects of AGEs with the naked eye. What is responsible for skin wrinkling? And for loss of skin flexibility? AGEs, it is believed, could be one culprit.

During AGE formation, sugars can combine with more than one protein, creating crosslinks between proteins. This increases the rigidity of tissues and decreases their elasticity. Alternately, AGE formation in collagen may disrupt the collagen network of cells, which could explain age-related arthritis and bone brittleness.

Similarly, the crosslinking effects of AGEs may be an underlying factor in many other diseases. For instance, the AGE content of skin collagen has been linked to diabetes-related risks. Scientists have suggested that AGE glycation makes the surface of LDL, the “bad cholesterol,” more ragged and thus more likely to stick to artery walls. Other studies have implicated AGEs in the triggering of receptors that stimulate growth factors and sustain plaque buildup.

AGEs may also lead to sight-related diseases, such as cataracts, by preventing cells from spreading properly, and to kidney disease, by playing a role in the formation of lesions. AGE formation in the blood vessels around neurons could lead to wall thickening, which is damaging to nerve tissue. And if AGEs form directly on the protein sheaths of nerve cells, the nerve cells’ covering may degrade and slow neuronal messages. The connection of AGEs to these diseases is bolstered by the fact that the effects listed above are especially common in diabetics, where high blood sugar levels increase the number of sugars available to undergo AGE formation.

Not all investigation into AGEs has implicated them in detrimental processes, however. Research has revealed the existence of inflammation receptors that recognize AGEs, opening the possibility that the molecules may be critical for biological processes. Yet why our body needs these receptors is still an open question.

The Challenges of Studying AGEs

Solving these mysteries of AGEs is likely to be a long process, as conducting research on the molecules can be difficult. We know the structures of only a fraction of the AGEs out there, which makes it even harder to assess accurately their properties and influences. Moreover, this makes it nearly impossible to assign potential correlations to specific molecules, as the effects may be due to new AGEs of which we are currently unaware.

Trying to assign a role in a disease to a specific AGE is a bit like being asked to find the source of obesity in New York, when the only information available is that fatty foods have been shown to correlated with obesity. What you do not know is which fatty foods contribute what to the problem.

Another obstacle in the investigation of AGEs is obtaining them. To study the structures and unique properties of each AGE, researchers need pure samples of the molecules. However, AGE formation is not very specific, as it is not directed by enzymes, and is quite slow. Despite all we have learned about AGE formation, like the special role arginine and lysine play in the process and the fact that certain amino acid sequences favor AGE formation, the most prevalent methods for obtaining AGE samples are still not much more advanced than isolating them from pretzels.

Moreover, once samples of AGEs are collected, they are difficult to separate. For example, a group of AGEs called hydroimidazolones has three structures that can arise from the same starting material. This structural similarity makes them difficult to separate by traditional means, which require significant differences in polarity or structure.

Spiegel Lab Tackles Synthesis

Yale Chemistry Professor David Spiegel is setting out to advance AGE research. His lab is synthesizing pure samples of several AGEs with known structures, including carboxymethyllysine (CML), hydroimidazolone, and glucosepane. Even more ambitiously, his lab is working on synthesizing these AGEs in protected forms, as amino acid monomers. These monomers can then be sequenced into short protein chains to observe AGEs at work in natural, biological contexts. When their investigation is complete, researchers around the world may gain access to pure samples of well-characterized AGEs.

The lab is already making significant progress in this endeavor. In only his first year at Yale, postdoctoral researcher Rendy Kartika has already synthesized pure samples of two of three hydroimidazolones. Taehan (Phil) Kim successfully synthesized CML, while Maria Noy and Alison Wendlandt are making headway on the bulky glucosepane.

The Wide Intrigue of AGEs

To Professor Spiegel, the problem of AGE synthesis is not one of just medical interest. Instead, he views it as an “intriguing challenge in its own right.” Spiegel finds it remarkable that these natural products have been around from the beginning of life, without any known enzymes to regulate their function or degradation. As no mechanisms for destroying AGEs are known, it is natural to ask ourselves: are AGEs completely bad?

The attachment of sugars to proteins is not unique to AGEs. In fact, modifying proteins with sugars is a trademark of eukaryotic cells. In fact, genetic mutations that impede an organism’s ability to glycosylate its proteins often result in severe developmental problems.

In the extracellular matrix, glycoproteins can form super complexes of molecular weight 2,000,000 (for reference, the average protein has a molecular weight of approximately 10,000). The sugars serve as markers that can identify a protein for delivery or destruction. The only difference is that these normal “glycations” (the technical term is glycosylation) are highly specific because enzymes direct them. Could AGEs have served primitive cellular functions before enzyme-directed glycosylations were possible?

Spiegel says these AGE structures simply “look like they’re biologically active.” If you break down the structure of an AGE, you find that it is a mixture of heterocycles (rings composed of both carbon and non-carbon atoms), straightchains, and multiple hydroxyl (-OH) groups. These are the same elements found in many biological molecules and approved drugs. Thus, it is conceivable that AGEs are biologically active as well. Furthermore, even if they are not, synthesizing AGEs could lead to insights for the production of drugs and other biologically active compounds.

Research on AGEs like that done in the Spiegel lab may lead to applications in many disciplines. For example, pure AGE samples have the potential to enhance the data quality of medical experiments, and these attempts at difficult syntheses may lead to valuable new synthesis methods. In addition, with pure AGE samples, we can begin exploring their chemical properties, which may very well lead us to a better understanding of AGE function in the body. As the mysteries of AGEs unfold, it is likely that so too will we move closer to solving mysteries in various fields of study.