Our bodies are highly efficient “glucose machines”, perfectly adapted to scarcity but challenged by today’s abundance of sugar. In this blog, we explore the role of glucose in metabolism, the impact of spikes and crashes, and how smarter dietary choices can help maintain more stable energy levels.
Carbohydrates are one of the most abundant class of molecules in nature. They are used by living organisms as energy storage, information transfer, a means for transportation, for protection, and the list goes on. This is thanks to the large amount of building blocks carbohydrates may have. In total, 27 natural monosaccharides exist, which can be joined together in different di-, oligo- or polysaccharides. For comparison, DNA is composed of only 4 different nucleotides (A, T, C, G) and the vast world of proteins is composed of 20 different amino acids. Both DNA and proteins offer enough combinatorial space to make up the entirety of life on earth.
Imagine the variety of carbohydrates that can be made! And to add an extra layer of complexity, while DNA and proteins follow strict rules in the linkages which bind amino acids or nucleotides, monosaccharides can bind to each other in a variety of different linkages! How can one even begin to study these complex molecules? Well, with a toolbox full of analytical methods, lots of collective experience and a bit of ingenuity.
Here we will examine some common methods in the carbohydrate analysis toolbox, what information do they provide, what are their limitations, and how they can work together to give a (close to!) full picture. Let’s work with a hypothetical carbohydrate and see what we can learn using our tools.
To start, let’s take a closer look at the monosaccharides in your carbohydrate. In a simple case, like beads on a necklace, the monosaccharides are all in a straight line connected by linkages; in a more complicated case, your carbohydrate might be a network of linkages. These linkages can be hydrolysed (cut) with acid releasing the individual beads for a closer look. However, the extent of hydrolysis can be tricky to predict. Is your carbohydrate a simple necklace and only a few cuts are necessary? Or is it a case of cutting a net? Through trial and error, we can determine when we achieve total hydrolysis, and our monomer beads are free for analysis. But hold on! What if you’ve been a bit too eager with your scissors? Some monomers are more delicate than others and end up breaking down during hydrolysis. This may compromise our results, delivering an incomplete picture of what beads are in our carbohydrate lattice. Thus, a delicate hand and plenty of knowledge and patience are needed.
Now that we know what monomers and how many are in the carbohydrate, it is time to understand how they are interconnected. We can take an enzymatic or a chemical approach. In the first case, enzymes that only cut specific linkages have been well studied. If we suspect our carbohydrate may have these linkages, we can unleash these enzymes and see if your carbohydrate has indeed been cut. These enzymes work well but can be a bit too case specific. For a more generalist approach we can try the chemical way. Methylation linkage analysis tags the unlinked carbon on a monomer then severs the link between each monomer. By examining the resulting pieces, we can determine how each monomer was linked to another. Although we may not know exactly in which position they were assembled, at least we know what type of linkages were present.
Finally, we would like to know the size of our carbohydrate. Luckily there is just the tool for that! With some caveats… Using size-exclusion chromatography, we can separate carbohydrates by size. Carbohydrates are run through sieve that catches smaller molecules and allows larger molecules to run through. So, whatever comes off the column first is larger, and what comes off later, the smaller carbohydrates. However, in this system, the shape of a carbohydrate will also affect how slow or fast it runs through the sieve, making something smaller with an awkward shape, for example, behave as if it was bigger than it is. Other techniques can somewhat distinguish between shapes, offering a clearer picture of your carbohydrate’s size.
If all has gone well, by now you should know by now the monomer composition, how they are linked to each other, and the rough size of your carbohydrate. But what is everything has not gone well? Well, that is where all the fun of carbohydrate analysis comes in. Drawing from knowledge, experience and a bit of ingenuity, each of these methods can be tailored to your particular carbohydrate. And your accumulated data so far can give you a pretty good idea of the molecule, and if needed, more tools can be added to the toolbox: Nuclear magnetic resonance (NMR), anion exchange chromatography, mass spectrometry, and the list goes on! And while these tools have advanced the collective knowledge of carbohydrate structures, many more structures are still being discovered and elucidated.
After reading this blog, you might feel dizzy with all the complex carbohydrate questions. This is where CarbExplore comes in, it’s literally in our name! Not only are we experts in carbohydrate analysis, but we also relish the challenge. So, if you are curious about your carbohydrate, leave the challenge to us.
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