Twenty-one IBS-D patients who came in every Monday were enrolled from the outpatient clinic in the Department of Gastroenterology and Hepatology of the First Affiliated Hospital of Sun Yat-sen University from September 2017 to November 2018, and 14 HCs were recruited by public advertising. All patients enrolled in this study fulfilled Rome Ⅳ diagnostic criteria for IBS and aged from 18 to 60. Patients were excluded if they had infectious gastroenteritis, organic gastrointestinal disease, previous abdominal surgery, lactose intolerance, metabolic diseases, human immunodeficiency virus infection and renal, cardiac or hepatic disease. Subjects taking any probiotics, prebiotics, antibiotics, or IBS prescription medications one month prior to baseline of our study were also excluded from our study. Age and gender matched HCs that they had no concomitant diseases, recurring GI symptoms, clinically significant abnormalities and medication taken.
13C labeled acetic acid( = 99%, isotopic purity), 13C labeled propionic acid ( = 99%, isotopic purity) and 13C labeled butyric acid ( = 99%, isotopic purity) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). A 0.9 M H2SO4 solution was prepared by diluting H2SO4 (98% purity) (Guangzhou, China). Water was deionized by using a MQ-water (Millipore, Bedford, USA). Sodium chloride (Guangzhou, China).
Fecal and serum samples
Twenty-one IBS-D patients and 14 HCs were asked not to take anything for at least 12 hours before their stool samples and peripheral venous blood were collected. Samples could be obtained in the morning. Since SCFAs are volatile and feces contain high concentrations of microbes. In order to keep the biological material in appropriate conditions after its collection, samples were stored at –80°C until to be analyzed. The fecal sample was immediately homogenized, and then stored. Blood was centrifuged (4000rpm, 15min) and the serum was collected and stored at −80°C.
Ten stool samples with a mass of 0.5g were separately added with 5mL of 125mg L-1 three 13C labeled acids, with 2g of sodium chloride (NaCl) and with 100 µL of a 0.9 M H2SO4 solution (pH = 2～3). Then the sample would be swirled for 5 minutes to homogenate. Finally, these vials were hermetically closed and submitted to test center in the south campus of Sun Yat-sen University to extract. Follow these steps, the additional concentration of the three labeled acids were varied several times until it was approximately equal to the target acid in the samples. The same procedure is used for pretreatment of serum samples except for deproteinization with methanol. All samples were sent to the south test center of Sun Yat-Sen University for testing.
Headspace solid-phase microextraction
Carboxen/polydimethylsiloxane (CAR/PDMS) 75 µm was applied to extract. The CAR/PDMS fibre gave best recoveries for the most volatile analytes like acetic and propionic acid . HS-SPME conditions were as follows: extraction temperature 60°C, extraction time 24 min and salt addition.
Gas chromatography–mass spectrometry
The carrier gas was helium (pressure 115kPa; flow 1.3ml min-1). Chromatographic separation was performed on a Supelcowax 10 fused-silica bonded-phase capillary column (30m×0.25mm; film thickness = 0.25μm; Supelco). GC oven temperature program was from 100℃ to 120℃ at 5℃ min-1 , then from 120℃to 150℃at 2℃ min-1, at last, from 150℃to 240℃ at 30℃ min-1 and the temperature should be1 min. The injector temperature was 250℃; the interface and the source temperatures were 280℃ and 200℃respectively. Electron impact mass spectra were recorded at 70eV ionisation energy (scan time, 0.2s; electron multiplier voltage, 700V) scanning the mass spectrometer from 15 to 550amu. Recorded mass spectra were compared with those stored in the National Institute of Standards and Technology (NIST) US Government library. Quantitative analysis was performed by measuring total ion current chromatographic peak areas. Firstly, the Thermo Xcalibur Roadmap software (Thermo Electron Corporation) was used to integrate the peaking areas of labeled acids and target acids in the tested sample. Then, the single point method was used for quantitative analysis of SCFAs, and concentration of target acids were calculated in the sample as follows: Am = Ai * Bm /Bi. where Am is the measured concentration of target acid in the sample and Ai is the peak area of the target acid. Bm and Bi represent measured concentration of 13C labeled acids and peak areas of 13C labeled acids in the sample solution injection volume. The amount of acetic, propionic and butyric acid in each sample was calculated by the above formula.
Evaluation of method performance
The chromatograms of SCFAs in feces and serum extracted are shown in figure1. As can be seen, the peaks of SCFAs were very well separated. Chromatograms of extracted feces and serum reflected the high enrichment and high selectivity for the SCFAs.
As shown in Figure1.The names of several acids with peaks in the chromatogram are determined in the mass spectrogram by the properties of the proton of the acid. The peak at about 7 min was identiﬁed as acetate, while the peak at about 9 min was identiﬁed as propionate and the peak of butyric acid at approximately 11 min. The acetic acid in serum was undetected, it could not be detected even after doubling serum concentration.
The repeatability and reproducibility of the method were expressed as the relative standard deviation (RSD) of peak areas of 13C labeled acid. And the precision was evaluated with RSD (%). The acceptable precision was <10%（RSD）. The repeatability and reproducibility of the system before each injection were obtained an RSD 2.36% ~ 6.29%. Then, according to the difference of the number of protons in the target SCFAs and the labeled SCFAs, the chromatogram of the target SCFAs and the labeled SCFAs were separated and calculated.
Statistical analysis was performed using the dedicated statistical software SPSS version 20.0 and Graph Prism version 7.0 (GraphPad software, Inc, La Jolla, CA, United States). The Student T-test was used for the comparisons between the groups. P values of < 0.05 were considered statistically significant.