Chapter 4 — Sample Types for Metabolomics

Metabolites in Blood

Human blood contains hundreds of metabolites that act as useful diagnostic tools for a person’s health. It may seem a trivial process to collect blood samples, but there are several important considerations to make when sampling and processing blood samples:

• Blood samples can be processed as plasma or serum. Plasma and serum are obtained from the liquid portion of blood once the cells are removed. Serum is the liquid portion after clotting has occurred, while plasma is the liquid portion remaining after an anticoagulant is added. Both sample types yield similar metabolomes,¹ but certain anticoagulants such as ethylenediaminetetraacetic acid (EDTA) or citrate,² can appear in plasma metabolomes. Heparin is less likely to interfere with mass spectrometry (MS) signals. Carefully consider which anticoagulant to use if you’re studying plasma and be sure you have a plan in place to remove background signal during data analysis, if necessary.
• Avoid hemolysis at all costs. Regardless of the type of blood being collected, researchers must strive to avoid the lysis of red blood cells (RBCs). When RBCs are lysed, hemoglobin and other intracellular components are released.³ These molecules can interfere with MS-based quantification of other blood metabolites.⁴
• Store samples at -80 °C. Samples should be stored as soon as possible at -80 °C after collection. They should also be stored as aliquots in separate sample tubes to minimize freeze-thaw cycles that can alter the blood metabolome.⁵

Metabolites in Urine

Urine consists of water-soluble molecules such as urea, ions, toxins, and other waste products that the kidneys extract from the bloodstream. Urine also has low cell counts, reducing biological noise in the urine metabolome.⁶ They are also easy to collect since urine can be obtained at home or in the laboratory. Despite these benefits, certain considerations need to be made when processing urine samples:

• High salt content. Urine samples may contain high salt concentrations that induce adduct formation during MS. The presence of multiple adducts together can reduce the sensitivity for detecting metabolites in samples.⁷ Sample extraction must therefore remove the interfering salts or their signals must be detected and removed during data analysis.⁸
• Sample dilution may be required. Some highly abundant metabolites, such as urea, can mask the MS signals of low-abundance metabolites. In such situations, diluting urine will increase the signal strength of other metabolites in the sample. Doing this step, however, requires a normalization step to account for differences in urine volume, water content, and water density before and after dilution.⁹
• Samples must be processed quickly. Fast processing times are essential for generating robust urine metabolomes. This is because urine metabolomes can change very quickly even when stored at temperatures as low as 4°C.¹⁰ If samples cannot be processed quickly, storage at -20°C or -80°C can retain urine metabolomes prior to processing.
• Other considerations. Cell removal should be considered since epithelial cells can be present at low abundances in urine. Additionally, while enzymes can be present in urine, adding an inhibitor is not recommended as they can obfuscate MS signals from endogenous molecules.¹¹

Metabolites in Feces

Fecal metabolomes provide a useful proxy for the activities of the gut microbiome and their links with host health, lifestyle, and genetics.¹² The intricate links with digestive and bodily health through the various axes such as the gut-brain axis¹³ make fecal metabolomes an appealing sample type to study. Studying fecal metabolomes also comes with a series of technical considerations that affect your processing protocols:

• Feces contain various classes of biomolecules. Although fecal matter is ~75% water, it also contains a wide range of biomolecules such as undigested organic matter, bacterial biomass, carbohydrates, fats, proteins, and organic gasses.
• Fecal matter is highly heterogeneous. Many factors can impact the appearance and chemical composition of fecal matter. These include diet, patient health status, medications, and host genetics.¹⁴ This complicates efforts to standardize extraction protocols from fecal matter.¹⁵
• Samples can be stored at room temperature. Fecal samples can be stored at room temperature for a few days with certain preservatives. Long-term storage, however, will require samples to be stored at -80°C.

Metabolites in Tissue

Studying tissue metabolomes provides a range of useful insights for biomedical research. Tissue biopsies are used to diagnose cancer, study organ physiology, and monitor disease status. Although tissues may be less complex than other sample types, processing them for metabolomics research also requires careful consideration:

• Quick processing times are essential. Cellular metabolism continues even after biopsies are collected.¹⁶ Therefore, samples should either be processed immediately or flash-frozen in liquid nitrogen to retain the tissue metabolome as close to in situ as possible.¹⁷ Alternatively, samples may be stored at -80°C before processing.
• Removing non-tissue contaminants. Tissue samples can contain connective tissue, fat, and residual blood that can obscure the tissue metabolome.¹⁸ Any extraction protocol with tissues must first remove these components through deionized water rinsing to prevent contamination.
• Extraction protocols depend on the tissue type. As is the case with many other sample types, multiple different organic solvents have been used to process tissue samples under the assumption that metabolomes are non-selective and reproducible. However, each tissue sample type may require unique extraction protocols,¹⁵ and while efforts have been made to standardize these protocols, more work needs to be done.

Other Sample Types for Metabolomics

While the four sample types described above are the most frequently studied in metabolomics research, metabolomics analysis can be performed on any sample type, providing a means to extract the metabolites has been developed. Other sample types and considerations for processing them include:

• Culture media: In vitro research is useful for studying cellular physiology, especially for bacterial cells. When comparing metabolomes between studies, researchers must consider the media employed in each study. The selection of media can have a substantial impact on the metabolomes being produced by the growing cells.¹⁸
• Soil: Soil is the medium that drives the world’s ecosystem. Stored as soil organic matter, soil metabolites can indicate soil type, biogeochemical processes, and effects of anthropogenic activity on environmental health.¹⁹ Typical soil extraction protocols fumigate the soil with chloroform vapors to release intracellular metabolites before extracting metabolites using high-salt extraction buffers.²⁰ Soil also contains organic volatile compounds that require a distinct extraction protocol to stabilize and characterize.²¹
• Water: Hundreds of new compound classes are being discovered in water, especially in marine waters.²² However, marine environments can be highly complex, depending on the aspects of the marine ecosystem being studied.²³ While typical extraction protocols with organic solvents work for water, these will have to be modified depending on the sets of metabolites being studied.

What’s Next?

Now that you understand the considerations to make when collecting different sample types for metabolomic analysis, we’ll take a deep dive into sample prep, storage, and transportation. Utmost care during these steps will ensure the highest quality metabolomes possible so you can extract actionable insights from your study.

References

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