Fecal short-chain fatty acids (SCFAs) assay

What Are Short-Chain Fatty Acids (SCFAs)?

Short-chain fatty acids (SCFAs) are the main metabolites produced by intestinal microorganisms fermenting dietary fiber, which mainly include acetic acid, propionic acid and butyric acid. They have important roles in maintaining intestinal health, regulating immunity and metabolism. Detection of SCFAs in feces is an important means to study the function of gut microorganisms and their relationship with host health.

For microbial communities, SCFAs are considered waste end products, but in anaerobic environments, they help maintain the redox balance in the gut.

The Role of SCFAs in Metabolism

Previous studies have shown that SCFAs play an important role in preventing and treating a range of metabolic syndromes, such as intestinal diseases and certain cancers. Clinical research suggests that SCFAs may have potential as an adjunctive therapy for immune-related intestinal diseases, such as Crohn’s disease, ulcerative colitis, and antibiotic-associated diarrhea. However, the mechanisms remain unclear and clinical efficacy has yet to be definitively established. This indicates that SCFAs effectively promote energy metabolism in mammals. Excessive energy intake and lack of physical exercise can lead to metabolic syndromes, including hypertension, obesity, dyslipidemia, and uncontrolled blood sugar. Epidemiological studies have shown that higher dietary fiber intake benefits body weight, food intake, and glucose homeostasis, while reducing the risk of metabolic disorders such as diabetes, cardiovascular diseases, irritable bowel syndrome, inflammatory bowel disease, and colon cancer. These benefits are closely related to the production of beneficial SCFAs through the fermentation of dietary fiber by gut bacteria.

The amount and type of dietary fiber significantly influence the composition of gut bacteria, thereby affecting the types and quantities of SCFAs produced. Typically, the in vivo production rate of SCFAs from different fibers correlates with the concentration of SCFAs in the gut. While cecal SCFA concentrations are available for most model organisms, information on in vivo production rates is difficult to obtain. In contrast, measuring cecal SCFA concentrations in the human gut is nearly impossible. Therefore, most studies estimate cecal and colonic metabolism by measuring fecal SCFA concentrations or through in vitro studies.

Applications of SCFAs

• Pathological Mechanism Research: SCFAs are valuable in researching the pathological mechanisms of gastrointestinal diseases and metabolic disorders and are being explored for their potential roles in liver conditions such as non-alcoholic fatty liver disease.
• Auxiliary Disease Diagnosis: While SCFA levels are being investigated as potential indicators for gastrointestinal diseases and certain metabolic disorders, their use as diagnostic markers for liver and gallbladder diseases is still under research.
• Drug Development and Treatment Monitoring: SCFAs are valuable in drug development and evaluating treatment efficacy.

Detection Methods

• Gas Chromatography (GC): High sensitivity and specificity, suitable for quantitative analysis of various SCFAs.
• Gas Chromatography-Mass Spectrometry (GC-MS): Combines the separation capability of GC with the identification power of MS, ideal for complex sample analysis.
• High-Performance Liquid Chromatography (HPLC): Suitable for detecting specific SCFAs.

Experimental Steps

1. Sample Collection and Preparation

Collection: Fresh fecal samples are collected and immediately placed in sterile containers.

Storage: Samples are stored at -80°C until analysis to avoid repeated freeze-thaw cycles.

Pretreatment: Weigh 100-200 mg of fecal sample, add phosphate-buffered saline (PBS) or deionized water, and homogenize thoroughly. Centrifuge (10,000 g, 10 minutes, 4°C) and collect the supernatant.

• SCFA Extraction

Acidification: Add concentrated hydrochloric acid or formic acid to the supernatant to adjust the pH to 2-3, converting SCFAs to their free acid forms.

Extraction: Add organic solvent (e.g., ether or ethyl acetate), mix thoroughly, centrifuge, and collect the organic phase.

Concentration: Evaporate the organic phase using nitrogen gas and redissolve in a small volume of solvent (e.g., methanol).

• Gas Chromatography Analysis

Instrument: Gas chromatograph equipped with a flame ionization detector (FID).

Column: Capillary column (e.g., DB-FFAP or HP-INNOWAX).

Carrier Gas: High-purity nitrogen or helium.

Temperature Program: Initial temperature: 50-60°C, hold for 1-2 minutes; ramp rate: 5-10°C/minute; final temperature: 200-250°C, hold for 5-10 minutes.

Injection: Inject 1-2 μL of sample using an autosampler or manual injection.

• Data Analysis

Standard Curve: Prepare a standard curve using known concentrations of SCFA standards (e.g., acetate, propionate, butyrate).

Quantification: Calculate SCFA concentrations by comparing sample peak areas to the standard curve.

CD BioGlyco can provide technical services for the detection of short-chain fatty acids in feces.

We offers sensitive, reliable, and accurate SCFA detection services. For more information, please feel free to contact us!