A recent study described develops methods for synthesizing and quantifying metal-induced glycation products, particularly in French fries, with phytic acid as a natural chelating agent (NCA). LCGC International spoke to Makan Golizeh, corresponding author of the paper inspired by this research, about the study and the team’s use of liquid chromatography-tandem mass spectrometry (LC–MS/MS) in their research.
Non-enzymatic glycation, which involves the reaction between a protein's amine group and a reducing sugar's carbonyl group, leads to advanced glycation end-products. Advanced glycation end-products (AGEs), formed in the body or ingested through processed and high-heat-cooked foods, contribute to oxidative stress, inflammation, and chronic diseases like diabetes complications and cardiovascular issues. Endogenous AGEs accumulate over time, damaging tissues, while dietary AGEs exacerbate these effects, especially in individuals with poor metabolic or kidney function. Reducing dietary AGE intake can help lower overall health risks. Metal ions, such as iron and copper, accelerate AGE formation through redox reactions, while chelators like ethylenediaminetetraacetic acid (EDTA) and natural chelating agents (NCAs) can inhibit this process. In food, glycation contributes to browning during cooking processes such as frying or baking. Factors like temperature, pH, moisture, and fat content influence AGE formation. High moisture cooking methods and acidic environments can reduce AGEs, improving food quality and nutritional value. A recent study described develops methods for synthesizing and quantifying metal-induced glycation products, particularly in French fries, with phytic acid as an NCA. LCGC International spoke to Makan Golizeh, corresponding author of the paper inspired by this research, about the study and the team’s use of liquid chromatography-tandem mass spectrometry (LC–MS/MS) in their research.
Your paper (1) evaluated the effect of phytic acid on the formulation of advanced glycation end-products (AGEs) in French fries. Briefly explain what these products are and why your work evaluating their effects is important.
Advanced glycation end-products (AGEs) are often chemically reactive compounds formed between amines and reactive carbonyls such as those present in carbohydrates. AGEs can undergo further chemical reactions, such as cross-linking and polymerization, altering the structure of nucleic acids and proteins, affecting their biological functions. AGEs have been shown to play a role in aging and the development of numerous diseases, such as atherosclerosis, diabetes, and neurodegenerative diseases. Moreover, AGEs are generally in low abundance and have a high molecular variety. Finding ways to accurately assess AGEs in biological mixtures is therefore important.
Why did you target French fries for your study as opposed to other types of food?
Potatoes contain high quantities of amino acids, carbohydrates, and iron, therefore having all the ingredients required for AGE formation. The latter, iron, as a prooxidant metal, catalyzes the oxidative reaction that produces AGEs. Although a slow reaction under physiological conditions, AGE formation significantly accelerates at elevated temperatures. French fries combine the ingredients and the high temperatures necessary to generate high amounts of AGEs, making it an excellent candidate for study.
Is phytic acid already in the potato, or is it created through a chemical reaction with the oil when frying the potato? If the latter, does the type of oil used to fry the potato (vegetable, peanut) effect the amount or quality of the acid generated? Is this acid generated if the potato is air-fried vs. deep-fried?
Phytic acid, also known as IP6, a natural metal-chelating agent abundant in grains, nuts, and seeds has been shown to have many health benefits. In this study, we have added phytic acid to potatoes prior to frying. Since phytic acid is a chemical compound, we also wanted to test the effect of one of its most prominent natural sources, rice bran, that contains up to 9% phytic acid, on metal-induced AGE formation in French fries. The choice of oil and frying method may have an effect on the quantities and types of AGEs formed in fried food; however, this was outside the scope of our study.
You state in your paper that AGEs are notoriously difficult to analyze. Why is this the case?
AGEs are typically difficult to analyze due to their extremely wide range of hydrophilicity, size, and variety of molecular structures, therefore no single method could be used to analyze all AGEs simultaneously in a mixture. For example, many low-molecular weight AGEs, such as monolysyl derivatives, are too hydrophilic to retain on conventional reverse-phase high performance liquid chromatography (HPLC) columns. On the other hand, heavier counterparts such as crosslinked protein aggregates are poorly soluble and thus incompatible with HPLC methods.
You used liquid chromatography-tandem mass spectrometry (LC–MS/MS) to identify glycation products within the foods tested. Why did you choose this analytical technique over other options?
While HPLC offers a high degree of separation power, mass spectrometry is a powerful technique for structural determination of low-abundance compounds. By combining the two, large numbers of soluble molecules could be characterized in a short amount of time with high accuracy and precision. Tandem MS also allows for structural confirmation of tentative molecular entities and, therefore, LC-MS/MS is an excellent tool for the analysis of complex matrices such as food samples.
Briefly summarize your findings that you discuss in your article, and the conclusions you came to after reviewing these findings.
Briefly, we have found that pH, temperature, and heavy metals, such as iron and copper, can affect the rate and direction of the AGE formation reaction. We also observed that chelating agents decrease the amounts of AGEs formed under experimental conditions and in food. Phytic acid, a natural chelating agent, showed promising results when added to the reaction mixture and to food before cooking. Throughout this study, we developed methods for laboratory synthesis of AGEs and their analysis by simple spectroscopic methods, as well as an LC–MS/MS approach to profile AGEs in complex mixtures, such as food samples. Moreover, we demonstrated that X-ray fluorescence (XRF) spectrometry is a reliable tool for quantitation of free metals in the presence of chelating agents. In conclusion, we showed that chelating agents can partially mitigate metal-induced AGE formation by moderating the ability of heavy metals to participate in redox reaction.
Do you anticipate similar results in using your technique to seek AGEs in other foods?
Yes, we do. While the overall performance of analytical methods largely depends on matrix composition and complexity, we anticipate our methods to be applicable to a wide range of food products. Our plans include the analysis of AGEs in other types of food products with high AGE content, such as bread, coffee, and pasteurized milk.
What difficulties did you encounter in your work, specifically sampling and analytical challenges?
Producing AGEs in the laboratory is slow and time-consuming. We tried to expedite the reaction using microwave irradiation; however, that was not as reproducible as the conventional incubation method. LC–MS/MS analysis is also relatively expensive, so we had to optimize our experiments before sending samples for analysis. Characterizing AGEs is not a trivial matter because of their wide variety of molecular structures making them susceptible to false discoveries and identity mismatches.
How did you process the LC–MS/MS data to obtain the results you were looking for?
A commercial computer program was utilized to acquire raw LC–MS/MS data and export them into a workable format, which was later manually processed by research students using Microsoft Excel and Python.
Were there any factors that might affect the accuracy of your findings?
Our laboratory experiments were limited to one amino acid (lysine) and one carbohydrate (glucose). By expanding this to other reactants, we could have a more realistic view of AGE formation and how metals may influence the chemistry of this reaction. Our food study was also similarly limited to russet potatoes. Since various types of potatoes have different constituents, our results may not be accurate across all potato types.
How do you imagine the results of your study can/will be applied?
Our aim was to provide insight into the mechanisms of AGE formation in human cells. The French fry study was an intermediary stage toward that aim. We were hoping to develop hypotheses and methods to facilitate the analysis of AGEs in much more complex samples, such as human cells. Others can use our methods to study AGEs in similar contexts.
Are there any next steps in this research?
We are currently working toward optimizing a human cell model to study metal-induced AGE formation using methods like those used in the French fry experiments. Alongside a research partner, we are formulating a targeted approach for the analysis of AGEs in human cells using affinity enrichment technologies. This will allow us to detect and quantify AGEs with very low abundances in complex biological mixtures, such as cell lysates.
References
1. Nobert, S.; Wolgien-Lowe, H.; Davis, T.; Paterson, E.; Wilson-Rawlins, T.; Golizeh,M. Assessing Metal-Induced Glycation in French Fries. Metallomics 2024, 17 (1), mfae059. DOI: 10.1093/mtomcs/mfae059
Makan Golizeh received his PhD in Analytical Chemistry from University of Quebec in Montreal (UQAM), in 2015, with a focus on LC–MS/MS analysis of reactive metabolites and their protein targets. His doctoral research led to a multitude of publications and conference presentations, as well as prestigious awards including the Mention of Honour of UQAM Faculty of Sciences, Governor General’s Academic Medal nomination, and the Michel Bertrand Award in Bioanalytical Mass Spectrometry. Makan completed postdoctoral training at Northeast Ohio Medical University in proteome dynamics of metabolic disorders and, in 2017, joined the Research Institute of McGill University Health Centre to work in the field of biomarker discovery of infectious diseases using top-down proteomics. In 2020, he accepted a faculty position at Concordia University of Edmonton, where he is currently Associate Professor of Chemistry and Department Chair of Environmental & Physical Sciences. Dr. Golizeh is a passionate bioanalytical chemist with an externally funded undergraduate-focused research program on heavy metals in environment and health. His research interest is to identify molecular profiles associated with diseases and pathogens in biological and environmental samples. Photo courtesy of Golizeh.
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