As we know, increased intake of dietary fiber may contribute towards a healthier diet. It has been studied that there are associations between increased dietary fiber intake and lower mortality from cardiovascular diseases and certain types of cancer. And the gut microflora in the large intestine, consisting of more than 400 different species, plays important physiological roles in human physiology and health. Microflora has the ability to produce hydrolytic enzymes that can digest some of the complex carbohydrates. When dietary fibers are fermented by the bacterial hydrolytic enzymes, the short chain fatty acids (SCFAs) are the main products. However, SCFAs may be generated from protein and amino acid decompositions as well.
Short chain fatty acids are defined as fatty acids with two to six carbon atoms. But the definition varies and the upper limit may range between five and seven carbons in length. Among SCFAs, three main types, acetic acid (C2), propionic acid (C3) and butyric acid (C4), makes up 95% of all SCFAs.
These SCFAs have a wide range of metabolic effects. They can be oxidized to provide energy and have also been shown to affect the immune system, colonic function, cholesterol metabolism, satiety and oxidative stress. In addition, A direct link between SCFAs (qualitatively and quantitatively) and some human pathological conditions, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), diarrhea and cancer have been proposed. So, there is no wonder that SCFA profiling has been a major topic in gut bacteria studies.
SCFAs have been measured in various biological materials such as blood plasma, serum, and feces. Moreover, SCFAs have been detected in different environmental samples, food, waste leachates and even in asphaltene. In the following parts, we will focus on the SCFAs analytical methods. Gas chromatography (GC) appears to be the most commonly used quantification method of fecal SCFAs despite having some disadvantages. Alternative methods include techniques related to liquid chromatography (LC), such as high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and capillary electrophoresis (CE).
Gas Chromatography (GC)
The principle of GC relies on a carrier gas that serves as a mobile phase where sample compounds are separated by differential interaction with the column stationary phase. The pretreatment is important for the successful detection of SCFAs by GC. There are various methods, such as ultrafiltration, centrifugation, acidification, or simple sample dilution, which has its own advantages or disadvantages.
The flame ionization detector (FID), is sensitive to molecules that are ionized in a hydrogen-air flame, including most carbon-containing compounds, and produces a current that varies proportionally to the amount of organic species in a sample, is the most used conventional detector for SCFAs detection in GC. In addition to a conventional detector, GC can be bound to MS, causing a better sensitivity and selectivity of the analysis. By using a GC-MS-MS instrument, it is available to quantitate SCFAs that are present in low concentrations in complex biological samples. GC-MS is an analytical technique, well suited for the analysis of SCFAs and other (longer) fatty acids. However, one critical step in the GC-MS analysis of FAs is their conversion into suitable volatile derivatives by derivatization (e.g., by alkylation or silylation). Some GC methods can cause a thermic degradation and structural modification of FAs during the methyl esterification process, or they can simply destroy the sample, disabling the possibility of its reanalysis.
High-Performance Liquid Chromatography (HPLC)
A great alternative to GC for SCFAs analysis is HPLC. Due to the higher pressure, the mobile phase carrying analytes travels and the small stationary phase particles with a larger area allow for a better interaction. The most commonly used technique is a reverse phase HPLC (RP-HPLC), where the stationary solid phase (column) is hydrophobic (non-polar) and the mobile liquid phase is hydrophilic (polar, watery). Like GC, a successful SCFAs analysis by HPLC needs suitable combinations of pretreatments, columns, running conditions and detectors. The greatest advantage of the HPLC over the GC technique is the use of lower running temperatures. And the resolved fatty acids are not destroyed during their detection, which enables further analyses to be performed.
In addition to GC and HPLC, there are some other methods, like nuclear magnetic resonance (NMR), Capillary Electrophoresis (CE), and enzymatic detection of SCFAs. In NMR, isotope cores 1H and 13C have been used for SCFAs studies in fecal samples. It enables the production of fast, stable and reproducible profiles, but the instrumentation cost and sensitivity are the serious drawbacks. CE, which has been used to detect SCFAs in different biological materials, is convenient in routine analysis due to its speed and minimal sample pretreatment procedure. However, the disadvantages of CE include low repeatability and reproducibility. Enzymatic detection of SCFAs depends on the spectrophotometric measurement of enzymatic products obtained from SCFAs as substrates. It can differentiate between optical isomers, so the lactate can be found in both D- and L-form.
Because of the connections between SCFAs, microbiota and metabolic diseases, a comprehensive understanding of SCFAs functional roles in the human body is essential. Therefore, there is a need for good and reliable analytical SCFA detection technologies. In summary, different SCFA analytical methods have advantages and limits. Because GC and HPLC have become standard and accessible in many laboratories. Moreover, GC-MS and HPLC-MS have pushed the limits of detection well below the actual concentrations of SCFAs found in feces. Therefore, GC-MS and HPLC-MS can be a suitable choice for low-concentration of fatty acids.
At Creative Proteomics, we have developed a professional platform for the Identification and quantification short chain fatty acids by GC-MS. The short chain fatty acids we can quantified include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid. If you want to know more, please visit our Short Chain Fatty Acids Analysis Service.
Reference:
- Primec M, Mičetić-Turk D, Langerholc T. Analysis of short-chain fatty acids in human feces: A scoping review. Analytical biochemistry, 2017, 526: 9-21.