
Short Chain Fatty Acids in Serum/Plasma and Fecal GC-MS Analysis Kit
Short-chain fatty acids (SCFAs) are organic acids generated by gut microbial fermentation of prebiotics, specifically undigested dietary carbohydrates, resistant starches, and dietary fibers. SCFAs are products of the metabolic effects of the microbiota and their interactions with food sources. SCFA levels are associated with obesity and diabetes: The gut microbiota appears to influence obesity and the development of type 1 and type 2 diabetes mellitus (T1D and T2D). Acetate, propionate, and butyrate—the most abundant SCFAs in our system—are produced by the gut microbiota in a roughly 3:1:1 ratio. Most are metabolized directly in colonocytes; the remainder is absorbed into the hepatic portal circulation, which provides energy to the liver, muscle, kidney, brain and heart. Absorbed into gut epithelia, acetate enters the tricarboxylic acid (TCA) cycle (also known as the citric acid cycle or the Krebs cycle) after being converted to acetyl CoA, and it produces ATP, cholesterol, and fatty acids. Propionate is the primary gluconeogenic substance in ruminants. Propionate is a (relatively small) substrate for gluconeogenesis in nonruminants, including humans, and is formed by the -oxidation of odd-chain and branched-chain fatty acids.
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Highlights
Total run time is 13.3 min.
Simultaneously analysis of SCFAs.
Consuming small volume of sample.
Long life span of GC column -
Parameters
Acetic acid, Propionic acid, Isobutyric acid, Butyric acid, Isovaleric acid, Valeric acid,
4-Methylvaleric acid, Hexanoic acid, Heptanoic acid -
Matrix
Serum/Plasma and Fecal
Sample Preparation
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Step 1Serum/Plasma Sample Preparation
Take 200 μl serum sample into a microcentrifuge tube -
Step 220 μl internal standard and 15 μl Reagent-1 are added, vortex for 10 seconds
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Step 3300 μl of Reagent-2 is added and hold in a shaker for 15 minutes. It is centrifuged at 10000 rpm for 10 minutes
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Step 4200 μl of the supernatant is taken into the HPLC vial
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Step 5300 μl of Reagent-2 is added to the lower phase again and vortexed for 10 seconds and centrifuged at 10000 rpm for 10 minutes
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Step 6Take 200 μl of the supernatant into the same HPLC vial and combine the supernatants
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Step 7Add 20 μl Reagent-3,vortex for 10 seconds.
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Step 8Incubated at 60°C for 30 minutes
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Step 9Injected into the HPLC vial at room conditions
Sample Preparation
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Step 1Fecal Sample Preparation
Weigh 30 mg of fecal sample into the centrifuge tube -
Step 21 ml of deionized water is added and shaken for 15 minutes in a mechanical mixer and kept at 2-8°C for 15 minutes
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Step 3Centrifuged at 10000rpm for 10 minutes
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Step 4Take 50 μl of the supernatant into a microcentrifuge tube
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Step 520 μl internal standard and 15 μl Reagent-1 are added, vortex for 10 seconds
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Step 6300 μl of Reagent-2 is added and hold in a shaker for 15 minutes. It is centrifuged at 10000 rpm for 10 minutes
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Step 7200 μl of the supernatant is taken into the HPLC vial
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Step 8300 μl of Reagent-2 is added to the lower phase again and vortexed for 10 seconds and centrifuged at 10000 rpm for 10 minutes
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Step 9Take 200 μl of the supernatant into the same HPLC vial and combine the supernatants.
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Step 10Add 20 μl Reagent-3,vortex for 10 seconds
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Step 11Incubated at 60°C for 30 minutes
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Step 12Injected into the HPLC vial at room conditions
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Chromatogram
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METHOD PERFORMANCE