Dec 16, 2025
Version 2
Short and Medium Chain Fatty Acid measurements of fecal, cecal, and serum samples V.2
- Blake Dirks1,
- Rosa Krajmalnik-Brown1
- 1Arizona State University
Protocol Citation: Blake Dirks, Rosa Krajmalnik-Brown 2025. Short and Medium Chain Fatty Acid measurements of fecal, cecal, and serum samples. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4wo7lmk/v2Version created by Anastasiya Moiseyenko
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 12, 2025
Last Modified: December 16, 2025
Protocol Integer ID: 234899
Keywords: vfa, volatile fatty acids, scfa, short chain fatty acids, hplc, scod, soluble chemical oxygen demand, fecal, cecal, serum, volatile fatty acids measurement, serum samples this method, serum sample, using hplc, acid content, mcfa, medium chain fatty acids, medium chain fatty acid measurement, acid content
Funders Acknowledgements:
Heritage Medical Research Institute
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Aligning Science Across Parkinson’s
Grant ID: ASAP-000375
Abstract
This method details how to measure short and medium chain fatty acid content of fecal, cecal, and serum samples obtained from mice using HPLC and normalizing to soluble chemical oxygen demand.
Guidelines
This protocol requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee and training for animal sacrifice by cervical dislocation.
This protocol has been approved by the California Institute of Technology’s Institutional Animal Care
and Use Committee (IACUC).
Materials
Eppendorf tubes
Serum gel microtubes
VFA standard mix
HPLC
COD kit
Spectrophotometer
Reactor block
Tissue collection
Using a sterile eppendorf tube, collect a fresh fecal pellet from animal and flash freeze on dry ice.
Sacrifice animal via cervical dislocation (CO2 or anesthetic use may alter or cause degradation of volatile fatty acids).
For serum, collect blood via cardiac puncture or from trunk into microtubes containing serum gel (such as Z-gel, Sarstedt AG & Co.) and place on ice for 30 minutes to allow clotting.
Once 30 minutes have passed, centrifuge tubes with serum gel at 10,000xg for 5
minutes and collect resulting serum into a separate eppendorf tube. Flash freeze on dry ice.
For cecal contents, dissect out cecum and squeeze contents out into sterile eppendorf tube. Flash freeze on dry ice.
Sample preparation
For fecal and cecal samples, add deionized water to the samples at a ratio of 1 mL of water to 100 mg of sample.
For serum samples, in order to precipitate any proteins in the serum, add 2.5 mM H2SO4 to samples at a 1:1 ratio.
Vortex serum samples briefly and place at 4°C for 20 minutes.
Vortex all samples at 3,200 rpm for 5 minutes
Centrifuge all samples at 13,000rpm for 15 minutes at 4°C.
Filter-sterilize all supernatants through a 0.22 μm syringe filter. Samples are now ready for HPLC analysis.
VFA measurements
Run samples on a machine, such as the Agilent 1260 Infinity II (Agilent, Santa Clara, California, USA) diode array detector, at 210nm with a Bio-Rad Aminex HPX-87H column (Cat. #: 1250095).
Column temperature should be at 65°C, with a 5 mM H2SO4 as a mobile phase at a flow rate of 0.6 mL/min run for 80 minutes.
Run a mixed standard, such as the 10 mM VFA mixed standard (Supelco Volatile
Free Acid Mix, Cat. #: CRM46975) containing acetate, formate, butyrate, isobutyrate, valerate,
isovalerate, propionate, caproate, isocaproate, and heptanoate, to generate a standard curve.
sCOD measurements
Measure sCOD (soluble chemical oxygen demand) using a HACH high-range (20 – 1500 mg COD/L) COD kit (Cat. #: 2565115). Normalizing all VFA measurements against the sCOD measured from the filtrate allows for accounting for differences in sample water content.
Add 100 μL of fecal or cecal sample filtrate, or 25 μL of serum sample filtrate to a HACH tube and adjust final volume to 2 mL with deionized water.
Digest samples on a reactor block, such as the HACH DRB200 Digital Reactor Block (Cat. #: LTV082.53.44001) according to the manufacturer’s recommendations.
Measure sCOD using a spectrophotometer such as the HACH DR6000 spectrophotometer (Cat. #: LPV441.99.00012).
Protocol references
Reynolds, M. C. et al. Delineating the Drivers and Functionality of Methanogenic Niches
within an Arid Landfill. Appl Environ Microbiol 88, e0243821 (2022).
Mohana Rangan, S. et al. Decoupling Fe0 Application and Bioaugmentation in Space and
Time Enables Microbial Reductive Dechlorination of Trichloroethene to Ethene: Evidence
from Soil Columns. Environ Sci Technol 57, 4167–4179 (2023).
Davis, T. L. et al. Chemical Oxygen Demand Can Be Converted to Gross Energy for Food
Items Using a Linear Regression Model. J Nutr 151, 445–453 (2021).
Dirks, B. et al. Methanogenesis associated with altered microbial production of short-chain
fatty acids and human-host metabolizable energy. ISME J 19, wraf103 (2025).
Corbin, K. D. et al. Host-diet-gut microbiome interactions influence human energy balance:
a randomized clinical trial. Nat Commun 14, 3161 (2023).