Metabolomics: measuring the molecules that power life

The central dogma of biology goes as follows: genetic information flows from DNA to RNA via transcription, and from RNA to protein via translation. That’s it. This is the fundamental framework for molecular biology.

But, this central dogma overlooks an important layer: molecules used in, and resulting from, metabolism, known as metabolites. While genes and proteins provide the blueprint and machinery of life, metabolites represent the chemical activities occurring inside cells at any given moment. They not only provide building blocks for all kinds of molecules and regulate energy balance, but are also required for modifications of proteins and epigenetic regulation at the DNA level. Due to these crucial roles, studying metabolites is essential to understand disease mechanisms, for biomarker discovery, and to identify therapeutic targets.

To study metabolites, we rely on mass spectrometry, a technique that converts molecules into charged particles and measures their mass-to-charge ratio with extremely high precision. In essence, mass spectrometry is a highly sophisticated (and expensive) molecular scale. If we know what we are looking for, it allows us to detect metabolites based on their precise molecular weight. You can see what one looks like in the picture below. For scale, I am 1.63 m tall: there is a certain irony in needing an instrument this large to study molecules this small!

The abundance paradox: why more isn’t always more

For those familiar with transcriptomics or proteomics, the initial analysis is often relatively straightforward: we compare condition A with condition B and generate beautiful volcano plots highlighting up- and downregulated genes or proteins (roughly: more and less active). Since few of us know the function of every gene and protein by heart, we then perform an extra analysis and eventually obtain a list of biological pathways that appear to be up- or downregulated. Suddenly our analysis is telling us, “Aha, ferroptosis (a type of programmed cell death dependent on iron) seems to be more active!”

Unfortunately, metabolites do not work quite like this.

Imagine that every metabolite is a lake and every enzymatic reaction connecting metabolites is a river connecting the lakes. If we look how much water is in a certain lake and realize “There is a lot more water compared to yesterday!” it is remarkably difficult to figure out why this is. Did something happen upstream, causing more water to flow into the lake? Or is the explanation hiding downstream: are branches or rocks blocking the water’s path? Maybe there even is an underground connection to the sea that we are not aware of and all of sudden more water from the sea is entering this lake. The only thing we can conclude is that there is more water compared to yesterday, but we cannot conclude anything on the dynamics of this system.

Metabolites behave in exactly the same way. If a certain metabolite is more abundant in condition B than in condition A, several explanations are possible: is there increased production? Or decreased consumption? Or is this metabolite being taken up from the extracellular environment? Or multiple processes may have changed simultaneously.

This is what we call the abundance paradox: metabolite abundances alone rarely reveal the underlying pathway dynamics. To understand pathway activity, we need a way to measure not only how much of a metabolite is present, but also where it comes from and where it goes.

Stable isotope tracing: the GPS tracker of molecular biology

How can we solve the abundance paradox? Let's return to our lake analogy. Imagine adding a brightly colored dye to one of the lakes. By following the dye, we could determine where the water flows, which lakes are connected, and even how quickly water moves through the system. What was previously a static snapshot suddenly becomes a movie of the ecosystem in motion.

In the lab we don’t rely on colored dyes, but nature brought us a very elegant solution which we call isotopes. Isotopes are atoms with the same number of protons, but a different number of neutrons. This difference in neutron number changes the mass of the atom without substantially altering its chemical behavior. Since mass spectrometers are exceptionally good at measuring tiny differences in mass, they allow us to distinguish molecules containing isotopes from their unlabeled counterparts.

Not all isotopes are the same. Some isotopes are radioactive and decay over time while emitting radiation. Others are stable and do not decay. Carbon provides a useful example. The most abundant form of carbon has 6 protons and 6 neutrons (12C), whereas the radioactive isotope of carbon has 8 neutrons (14C) and the stable isotope 7 neutrons (13C). Both radioactive and stable isotopes can be used to study metabolism, but stable isotopes are particularly attractive because they are harmless and can reveal detailed information about pathway activity and nutrient utilization.

Time for an illustration with some sugar! Glucose is one of the major carbon sources in mammalian cells and it feeds into a variety of pathways with fancy names, such as glycolysis, the pentose phosphate pathway, serine synthesis pathway, and the Krebs cycle. Suppose we replace all six carbon atoms in glucose with ¹³C atoms, generating uniformly labeled or U-¹³C-glucose. Cells can take up U-¹³C-glucose exactly as they would normal glucose and metabolize it through the same pathways. The difference is that we can now follow the labeled carbon atoms throughout the metabolic network. For every metabolite that we measure, we can determine not only how much of that metabolite is present, but also what fraction was synthesized from the labeled glucose. This quantity is known as the fractional contribution.

But we can get even more information: we can also determine how many labeled carbons a metabolite contains. A metabolite may contain zero, one, two, or more ¹³C atoms, producing a collection of labeled forms known as isotopologues. These isotopologue patterns often reveal which pathways were used to generate the metabolite, which is particularly interesting for metabolites that can be generated through multiple biochemical routes.

In even more complex situations, a metabolite may contain the same number of labeled atoms but differ in the location of those labels within the molecule. These distinct labeling arrangements are known as isotopomers. Because different metabolic routes can generate different isotopomer patterns, these measurements can provide even deeper insight into pathway architecture and carbon flow.

Together, metabolite abundances, fractional contributions, isotopologue distributions, and isotopomer patterns transform metabolomics from a static measurement into a dynamic analysis of metabolic pathway activity: not just a static photo of the lake ecosystem, but a detailed 3D render in which we can see the water flow. Remarkably, all of this information can be obtained simply by replacing naturally occurring atoms with their stable isotope counterparts. Amazing, right?

Isotopes aplenty, or: more than carbon

Carbon is not the only element that can be traced. Stable isotopes are also available for nitrogen (¹⁵N), hydrogen (²H or deuterium), oxygen (¹⁸O), and several other biologically relevant elements.

We can even combine multiple tracers in a single experiment. For example, glutamine is often labeled with both ¹³C and ¹⁵N to simultaneously track carbon and nitrogen metabolism. The possibilities become even more powerful when multiple nutrients are labeled in parallel. For instance, we could supply ¹³C-labeled glucose together with ¹⁵N-labeled glutamine, allowing both carbon and nitrogen metabolism to be tracked at the same time. The possibilities are endless!

However, one important rule is that two nutrients should never carry the same isotope label on the same element. While the mass spectrometer can accurately detect the extra mass contributed by a stable isotope, it cannot determine which nutrient originally carried that label. For example, if both glucose and glutamine are supplied as ¹³C-labeled tracers, the instrument can detect ¹³C atoms appearing in downstream metabolites, but it cannot distinguish whether those ¹³C atoms originated from glucose or glutamine.

Importantly, stable isotope tracing is not limited to cultured cells. The same principles can be applied to virtually any living system, including organoids, tissue slices, perfused organs, tumor explants, animal models, and even human studies. Wherever metabolism occurs, stable isotope tracing helps us track where atoms come from, where they go, and which routes they take along the way.

The flow of life

Metabolomics has transformed our ability to measure the small molecules that drive life. By providing a snapshot of hundreds of metabolites simultaneously, it has become an indispensable tool for studying health, disease, and cellular function. However, as powerful as these measurements are, metabolite abundances alone only tell part of the story.

A metabolite is much more than a number on a graph. Its abundance reflects the balance between production, consumption, transport, and storage, making it difficult to make conclusions about pathway dynamics from abundances alone. Stable isotope tracing overcomes this limitation by allowing us to follow the journey of atoms through metabolic networks. In modern metabolism research, it is no longer just a powerful tool, it is often the difference between observing biology and truly understanding it.

Shauni Loopmans

Expert Metabolism & Data Interpretation, VIB Metabolomics Core - Leuven

• shauni.loopmans@kuleuven.be