Free Fatty Acids Signature in Human Intestinal Disorders: Significant Association between Butyric Acid and Celiac Disease

Altered circulating levels of free fatty acids (FFAs), namely short chain fatty acids (SCFAs), medium chain fatty acids (MCFAs), and long chain fatty acids (LCFAs), are associated with metabolic, gastrointestinal, and malignant diseases. Hence, we compared the serum FFA profile of patients with celiac disease (CD), adenomatous polyposis (AP), and colorectal cancer (CRC) to healthy controls (HC). We enrolled 44 patients (19 CRC, 9 AP, 16 CD) and 16 HC. We performed a quantitative FFA evaluation with the gas chromatography–mass spectrometry method (GC–MS), and we performed Dirichlet-multinomial regression in order to highlight disease-specific FFA signature. HC showed a different composition of FFAs than CRC, AP, and CD patients. Furthermore, the partial least squares discriminant analysis (PLS-DA) confirmed perfect overlap between the CRC and AP patients and separation of HC from the diseased groups. The Dirichlet-multinomial regression identified only strong positive association between CD and butyric acid. Moreover, CD patients showed significant interactions with age, BMI, and gender. In addition, among patients with the same age and BMI, being male compared to being female implies a decrease of the CD effect on the (log) prevalence of butyric acid in FFA composition. Our data support GC–MS as a suitable method for the concurrent analysis of circulating SCFAs, MCFAs, and LCFAs in different gastrointestinal diseases. Furthermore, and notably, we suggest for the first time that butyric acid could represent a potential biomarker for CD screening.


S2. GC-MS method
The FFAs in the samples were analyzed as free acid form using an Agilent J&W DB-FFAP column 30 m length, 0.25 mm internal diameter and 0.25 m of film thickness by using the oven temperatures' program, as follows: initial temperature of 50 °C for 1 min, then it was increased to 150 °C at 30 °C/min, finally grow up to 250 °C at 20 °C/min was held for 6.67 min. A 1 µL aliquot of extracted sample was injected in splitless mode (splitless time 1 min) at 250°C, while the transfer line temperature was 280°C. The used carrier gas was helium and its flow rate maintained at 1 mL/min for the whole run time. The MS acquisition was carried out in single ion monitoring (SIM) by apply a proper dwell time (20 ms for each ion monitored) to guarantee an acquisition frequency of 4 cycle/s. The quantitative determination of FFAs in each sample was carried out by the ratio between the area abundance of the analytes and the area abundance of respective labeled internal standard (isotopic dilution method). The value of this ratio was named Peak Area Ratio (PAR) and it was used as abundance of each analyte in the quantitative evaluation. The ionic FFAs' signals and the reference internal standards used for the quantitation of each FFAs were reported in the Table S1. Retention times (Rt) and ionic signals used for quali-quantitation of FFAs and relative ISTD used.
The GC-MS profiles from the samples showed five unknown peaks (Rt 8.75,9.64,10.67,12.11 and 14.19 min respectively), with the characteristic fragmentation of carboxylic acids. By considering the selectivity factor of the chromatographic system used toward the carboxylic acids and the relative abundances of the characteristic fragmented ions, these unknown peaks could be as follows: -

S2.1 Standard solutions and calibration levels
The stock solutions of each analyte and each ISTD were prepared in mQ water at 50 mg/ mL -1 and stored at 4 °C.
Since the quantity of each FFA in the samples could be different, distinct concentration ranges of each analyte were defined. Therefore, to easily build up these calibration levels, two working mixtures of analytes (Mix 1 and Mix 2) and a mixture of ISTDs in 10 mM NaHCO3 solution were prepared. The compositions and the concentrations of these mixtures are reported in the Table S2.  while the other acids are 0.25, 0.50, 1.00, 2.50 and 5.00 μg/mL.

S2.2 Calibration curves
Calibration curves of analytes were obtained by plotting the peak area ratios (PAR), between quantitation ions of each analyte and relative ISTD, versus the nominal concentration of the calibration solution. A linear regression analysis was applied to obtain the best fitting function between the calibration points.
In order to obtain reliable limit of detection (LOD) and limit of quantitation (LOQ) values, the standard deviation of response and slope approach was employed. The estimated standard deviations of responses of each analyte were obtained by the calculated standard deviation of y-intercepts (SDY-I) of regression lines.
The obtained linear regressions coefficients, the r-squared (R2) and the estimated LOD values for each analyte are reported in the Table S3. Table S3. Linear regressions data, R-squared and LOD values obtained for each analyte.
Concerning the quantitative evaluation of the acids not included in Table S3, it was use the calibration parameters obtained from the nonanoic acid.