Supplementary Information for
A targeted metabolomic protocol for short-chain fatty acids and
branched-chain amino acids
Xiaojiao Zheng, Yunping Qiu, Wei Zhong, Sarah Baxter, Mingming Su, Qiong Li, Guoxiang
Xie, Brandon M. Ore, Shanlei Qiao, Melanie D. Spencer, Steven H. Zeisel, Zhanxiang Zhou,
Aihua Zhao*, Wei Jia*
1
Development of Biological Sample Preparation
As derivatization solvents optimization, the volumes of water, propanol (PrOH) and pyridine (Py)
should be kept as 800, 300 and 200 µL, respectively. Meanwhile, the pH value 8 provided optimal
derivatization efficiency.
Feces Sample. Extraction solvents were first investigated. Water, water/PrOH (v/v = 2:1),
water/PrOH (v/v = 1:1) were investigated. The solvents were selected with the consideration that very
short chain fatty acids and BCAAs are well water soluble, while the water solubility reduced with the
carbon chain increased, so that pure water as well as water mixed with organic solvent were
investigated. The pH were investigated by 0.02 M, 0.01 M HCl and 0.02 M, 0.01 M NaOH solvents.
As the pH had impact on derivatization efficiency, all the sample supernatants were neutralized with
HCl or aqueous NaOH after extraction. The results are presented in Fig. 1S. The heat-map on the left
shows that most of water extraction solvents with pH equal to 7-8 provide better extraction efficiency.
One more experiment was conducted to investigate the pH adjusted by different basic solvents, 0.005
M, 0.01 M aqueous NaOH or Na2CO3. The results are presented in the right heat-map, showing that
0.005 M aqueous NaOH provides the optimal extraction efficiency for feces. The pH value of
derivatization system was 8 after 500 µL sample extraction was added, which also provided optimal
derivatization efficiency.
Urine and Plasma. Urine and plasma are water-based biological fluids. They were directly
derivatized without extraction procedures. Aqueous NaOH solution was directly added to reaction
system to adjust pH to 8. Different volumes of urine and plasma were investigated, including 100, 200,
300, 400 and 500 µL. Both urine and plasma were determined as 300 µL, since most of SCFAs and
BCAAs could be detected.
2
Supplementary Fig. 1. The optimization of extraction solvent for feces sample. The color in each cell
presents the intensity compared to those in the other methods of the same compound. (white =
medium intensity, red = higher intensity, blue = lower intensity)
3
Derivatization Procedures Optimization
Supplementary Fig. 2. Derivatization procedures optimization with two one-step derivatizations and
three two-step derivatizations. H indicates the step of hexane extraction. B indicates the step of adding
aqueous NaOH to adjust pH 9-10.
4
Derivatives Extraction Solvent Optimization
Supplementary Table 1. The extraction efficiency of two extraction methods
Extraction efficiency of
one-step extraction (%)
Extraction efficiency of
two-step extraction (%)
Acetic acid
88.50
98.09
Propionic acid
86.02
96.55
Isobutyric acid
90.18
98.29
Butyric acid
90.13
98.45
2-Mehtylbutyric acid
90.96
100.00
Isovaleric acid
91.31
100.00
Valeric acid
99.89
100.00
Caproic acid
93.30
100.00
Heptanoic acid
100.00
100.00
Valine
89.18
100.00
Leucine
91.47
99.34
Isoleucine
91.52
99.32
Compounds
5
The Weight of Feces Sample Determination
Different weights of feces samples, 10, 25, 50, 75, 100, and 150 mg, were evaluated with the same
extraction and derivatization methods. The result showed that the concentrations of 2-methylbutyric
acid and isovaleric acid in 10 mg of feces sample were just around the LOD. All of the compounds
can be detected from 25mg to 150 mg. The calibration equation of each compounds showed good
linearity with R2 more than 0.99. As a result, the weight of samples ranging from 25 mg to 150 mg is
acceptable in our method.
Supplementary Table 2. Linearity of each compound in the feces samples from 25 mg to 150 mg.
Compounds
Acetic acid
Calibration Equation
r2
y = 0.0065x + 0.0332
Propionic acid
y = 0.0026x + 0.1352
0.9971
0.9972
Isobutyric acid
y = 0.0004x - 0.0017
Butyric acid
y = 0.003x - 0.018
0.9973
0.997
2-Methylbutyric acid
y = 0.0003x - 0.0008
0.9959
Isovaleric acid
y = 0.0002x - 0.0016
0.9983
Valeric acid
y = 0.0004x - 0.0024
0.9963
Caproic acid
y = 0.0008x - 0.0026
0.9969
Heptanoic acid
y = 0.0001x - 0.001
0.9985
Valine
y = 0.0053x - 0.0557
0.9942
Leucine
y = 0.011x - 0.1046
0.9976
Isoleucine
y = 0.0042x - 0.0462
0.9935
6
Supplementary Table 3. Derivatization efficiency and recovery
Compound
Derivatization Efficiency (%)
Low
Middle
Compound
Recovery (%)
High
Low
Mean
RSD
Mean
RSD
Mean
RSD
Acetic acid
109.76
0.85
98.59
7.55
103.98
1.68
Propionic acid
106.15
0.66
97.11
7.82
101.98
Isobutyric acid
103.68
1.07
94.34
8.60
Butyric acid
108.39
1.79
95.34
Isovaleric acid
106.34
2.04
Caproic acid
104.24
6.05
Middle
High
Mean
RSD
Mean
RSD
Mean
RSD
Acetic acid-d4
90.03
1.42
98.13
2.38
94.39
2.40
0.89
Propionic acid-d2
84.11
0.28
94.83
2.36
96.5
3.44
90.06
0.97
Isobutyric acid-d3
89.3
3.91
98.00
0.57
98.04
2.90
7.76
99.96
1.51
Butyric acid-d2
104.03
9.24
98.73
2.14
96.82
3.32
100.7
5.51
110.1
2.15
Valeric acid-d9
95.78
1.96
97.94
1.86
94.92
2.43
95.73
7.17
101.57
2.53
Caproic acid-d3
98.03
4.05
96.95
2.51
94.6
0.57
Heptanoic acid-d7
110.86
4.60
118.79
0.81
112.8
1.92
Valine-d8
87.82
11.03
106.42
1.61
106.88
1.81
Leucine-d10
101.76
11.83
110.61
1.91
106.94
2.58
7
Supplementary Fig. 3. Typical GC-MS chromatography of standard mixture (A) and fecal sample (B). 1. acetic
acid, 2. propionic acid, 3. isobutyric acid, 4. butyric acid, 5. 2-methylbutyric acid, 6. isovaleric acid, 7. valeric
acid, 8. 2-methylpentanoic acid, 9. 3-methylpentanoic acid, 10. isocaproic acid, 11. caproic acid-d6 (internal
standard), 12. caproic acid, 13. 2-methylhexanoic acid, 14. 4-methylhexanoic acid, 15. heptanoic acid, 16. valine,
17. leucine, 18. isoleucine.
8
Supplementary Fig. 4. The stability of each compound after derivatization. The three bars for each compound
represent the RSDs of peak areas detected within 2, 4, 7 days under three storage conditions: -20 ºC (top), room
temperature (23-25 ºC) (middle), 12-h room temperature/12-h -20 ºC cycle (bottom).
9
Application One: Animal Study
The animal studies were approved by the Institutional Animal Care and Use Committee, and
performed following the guidelines at the Center for Laboratory Animals, David H. Murdock
Institution (NC). Sprague Dawley male rats (16-18 weeks) were purchased from Charles River
Laboratories (Wilmington, MA). All rats were housed in a specific-pathogen-free (SPF) environment
under a controlled condition of 12 h light/12 h dark cycle at 20-22 oC and 45 ± 5% humidity. The
experimental rats were randomly separated into three groups (n = 8 in each group): 1) a “corn oil
group” that ingested 35% corn oil in the Lieber-DeCarli liquid diet; 2) a “corn oil/ethanol” group that
ingested the Lieber-DeCarli liquid diet containing 35% corn oil and 7% ethanol and 3) a “medium
chain fatty acid (MCFAs)/ethanol” group that ingested the Lieber-DeCarli liquid diet containing 35%
MCFAs and 7% ethanol. Fecal samples were collected after 15-day intervention and stored
immediately at -80 oC prior to metabolite extraction.
Application Two: Clinical Study
Healthy females (n=15) were recruited and provided with informed consent. Inclusion was
contingent upon a good state of health, age of 18-70, body mass indices of 18-34, and no history of
chronic system disease determined by physical examination and standard clinical laboratory tests.
Individuals using drugs or medications during the previous 3 months were excluded. During the study,
all participants were fed control diets for ten days to minimize the dietary-induced metabolism
variations. Morning urine samples and fecal samples were collected and immediately frozen at -80 oC.
Whole blood was collected into commercially available anticoagulant-treated tubes. Cells were
removed by centrifugation for 10 min at 3,000 g to obtain plasma. The plasma samples were
immediately transferred to a clean tube and stored at 80oC.
10
Supplementary Fig. 5. The discrimination of quantitative SCFAs and BCAAs in different groups shown by
heat-map and partial least squares-discriminant analysis (PLS-DA) plot. Each cell in the heat-map represents the
fold change of a particular metabolite in two different groups (red = higher concentration, blue = lower
concentration).
11