Fecal SCFA content was determined by gas chromatography. Chromatographic
analysis was carried out using a Shimadzu GC14-A system with a flame ionization
detector (FID) (Shimadzu Corp, Kyoto, Japan). Fused silica capillary columns 30m
× 0.25 mm coated with 0.25um film thickness were used (Nukol™
for the volatile acids and SPB™-1000 for the nonvolatile acids (Supelco
Analytical, Bellefonte, PA). Nitrogen was used as the carrier gas. The oven
temperature was 170°C and the FID and injection port was set to
225°C. The injected sample volume was 2 µL and the run time for
each analysis was 10 minutes. The chromatograms and data integration was carried
out using a Shimadzu C-R5A Chromatopac. A volatile acid mix containing 10 mM of
acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic,
caproic, and heptanoic acids was used (Matreya, Pleasant Gap, PA). A
non-volatile acid mix containing 10 mM of pyruvic and lactic and 5 mM of
oxalacetic, oxalic, methy malonic, malonic, fumaric, and succinic was used
(Matreya, Pleasant Gap, PA). A standard stock solution containing 1%
2-methyl pentanoic acid (Sigma-Aldrich, St. Louis, MO) was prepared as an
internal standard control for the volatile acid extractions. A standard stock
solution containing 50 mM benzoic acid (Sigma-Aldrich, St. Louis, MO) was
prepared as an internal standard control for the non-volatile acid
extractions.
Samples were kept frozen at −80°C until analysis. The
samples were removed from the freezer and 1,200µL of water was added to
each thawed sample. The samples were vortexed for 1 minute until the material
was homogenized. The pH of the suspension was adjusted to 2–3 by adding
50 µL of 50% sulfuric acid. The acidified samples were kept at
room temperature for 5 minutes and vortexed briefly every minute. The samples
were centrifuged for 10 minutes at 5,000g. 500 µL of the clear
supernatant was transferred into two tubes for further processing. For the
volatile extraction 50 µL of the internal standard (1% 2-methyl
pentanoic acid solution) and 500 µL of ethyl ether anhydrous were added.
The tubes were vortexed for 30 seconds and then centrifuged at 5,000g for 10
minutes. 1 µL of the upper ether layer was injected into the
chromatogram for analysis. For the nonvolatile extraction 50 µL of the
internal standard (50 mM benzoic acid solution) and 500 µL of boron
trifluoride-methanol solution (Sigma-Aldrich St. Louis, MO) were added to each
tube. These tubes were incubated overnight at room temperature. 1 mL of water
and 500 µL of chloroform were added to each tube. The tubes were
vortexed for 30 seconds and then centrifuged at 5,000g for 10 minutes. 1
µL of the lower chloroform layer was injected into the chromatogram for
analysis. 500 µL of each standard mix was used and the extracts prepared
as described for the samples. The retention times and peak heights of the acids
in the standard mix were used as references for the sample unknowns. These acids
were identified by their specific retention times and the concentrations
determined and expressed as mM concentrations per gram of sample.
analysis was carried out using a Shimadzu GC14-A system with a flame ionization
detector (FID) (Shimadzu Corp, Kyoto, Japan). Fused silica capillary columns 30m
× 0.25 mm coated with 0.25um film thickness were used (Nukol™
for the volatile acids and SPB™-1000 for the nonvolatile acids (Supelco
Analytical, Bellefonte, PA). Nitrogen was used as the carrier gas. The oven
temperature was 170°C and the FID and injection port was set to
225°C. The injected sample volume was 2 µL and the run time for
each analysis was 10 minutes. The chromatograms and data integration was carried
out using a Shimadzu C-R5A Chromatopac. A volatile acid mix containing 10 mM of
acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic,
caproic, and heptanoic acids was used (Matreya, Pleasant Gap, PA). A
non-volatile acid mix containing 10 mM of pyruvic and lactic and 5 mM of
oxalacetic, oxalic, methy malonic, malonic, fumaric, and succinic was used
(Matreya, Pleasant Gap, PA). A standard stock solution containing 1%
2-methyl pentanoic acid (Sigma-Aldrich, St. Louis, MO) was prepared as an
internal standard control for the volatile acid extractions. A standard stock
solution containing 50 mM benzoic acid (Sigma-Aldrich, St. Louis, MO) was
prepared as an internal standard control for the non-volatile acid
extractions.
Samples were kept frozen at −80°C until analysis. The
samples were removed from the freezer and 1,200µL of water was added to
each thawed sample. The samples were vortexed for 1 minute until the material
was homogenized. The pH of the suspension was adjusted to 2–3 by adding
50 µL of 50% sulfuric acid. The acidified samples were kept at
room temperature for 5 minutes and vortexed briefly every minute. The samples
were centrifuged for 10 minutes at 5,000g. 500 µL of the clear
supernatant was transferred into two tubes for further processing. For the
volatile extraction 50 µL of the internal standard (1% 2-methyl
pentanoic acid solution) and 500 µL of ethyl ether anhydrous were added.
The tubes were vortexed for 30 seconds and then centrifuged at 5,000g for 10
minutes. 1 µL of the upper ether layer was injected into the
chromatogram for analysis. For the nonvolatile extraction 50 µL of the
internal standard (50 mM benzoic acid solution) and 500 µL of boron
trifluoride-methanol solution (Sigma-Aldrich St. Louis, MO) were added to each
tube. These tubes were incubated overnight at room temperature. 1 mL of water
and 500 µL of chloroform were added to each tube. The tubes were
vortexed for 30 seconds and then centrifuged at 5,000g for 10 minutes. 1
µL of the lower chloroform layer was injected into the chromatogram for
analysis. 500 µL of each standard mix was used and the extracts prepared
as described for the samples. The retention times and peak heights of the acids
in the standard mix were used as references for the sample unknowns. These acids
were identified by their specific retention times and the concentrations
determined and expressed as mM concentrations per gram of sample.