Evaluation and comparison of short chain fatty acids composition in gut diseases

Table 1 Clinical characteristics of patients

ID patients	State	Number	Male/female ratio, n (%)	Age, median (IQR)	BMI, median (IQR)
HC	healthy control	16	7 (14/2)	46 (9)	23.8 (1.5)
CD	celiac disease	16	0.5 (6/12)	35.5 (21)	22.1 (3.7)
AP	adenomatous polyposis	9	1.2 (5/4)	68 (28)	24.9 (3.9)
CRC	colorectal cancer	19	8.5 (17.2)	80 (13.5)	24.5 (4.5)

HC: Healthy control; CD: Celiac disease; AP: Adenomatous polyposis; CRC: Colorectal cancer; BMI : Body mass index; IQR: Interquartile range.

Table 2 Retention times, ionic signals, internal standards for each short chain fatty acid

SCFA	Rt(min)	Quan. Ion(m/z)	Qual ion(m/z)	ISTD[quant. ion (m/z)]
Acetic	5.65	60	-	[2H3]Acetic[63]
Propionic	6.08	74	73	[2H3]Propionic[77]
iso-Butyric	6.22	73	88	[2H7]iso-Butyric[77]
Butyric	6.53	60	73	[2H3]Propionic[77]
iso-Valeric	6.73	60	87	[2H9]iso-Valeric[63]
Valeric	7.08	60	87	[2H9]iso-Valeric[63]

SCFA: Short chain fatty acid; Rt: Retention time; ISTD: Internal standard.

Table 3 Compositions and concentrations of standard and internal standards mixtures

Mixt-ure	Acetic ac. (µg/mL)	Propionic ac. (µg/mL)	Butyric ac. (µg/mL)	isoButyric ac. (µg/mL)	Valeric ac. (µg/mL)	isoValeric ac. (µg/mL)
Mix 1	10000	5000	5000	1000	1000	1000
Mix 2	1000	500	500	100	100	100
	[2H3]Acetic (µg/mL)	[2H3]Propionic (µg/mL)		[2H7]iso-Butyric (µg/mL)		[2H9]iso-Valeric (µg/mL)
ISTDs	1000	500		100		100

ISTDs: Internal standards

Table 4 Concentrations of each analyte in the calibration levels

Calibration levels	Acetic ac. (g/mL)	Propionic ac. (g/mL)	Butyric ac. (g/mL)	isoButyric ac. (g/mL)	Valeric ac. (g/mL)	isoValeric ac. (g/mL)
1	10.0	5.0	5.0	1.0	1.0	1.0
2	20.0	12.5	12.5	2.5	2.5	2.5
3	50.0	25.0	25.0	5.0	5.0	5.0
4	100.0	50.0	50.0	10.0	10.0	10.0
5	250.0	125.0	125.0	25.0	25.0	25.0

Table 5 Linear regressions data, R-squared and limit of detection values obtained for each analyte

Compound	Slope PAR (µg/mL)	Intercept PAR (µg/mL)	R2	LOD SDY-I (µg/mL)
Acetic	0.027	- 0.025	0.996	11.3
Propionic	0.088	- 0.142	0.996	5.9
Butyric	0.213	- 0.552	0.993	7.9
Isobutyric	0.357	- 0.153	0.994	1.4
Valeric	0.406	- 0.297	0.993	1.6
Isovaleric	0.459	- 0.327	0.991	1.7

PAR: Peak area ratios; R²: r-squared; LOD: Limit of detection; SDY-I: Standard deviation of y-intercepts.

Table 6 Median ± standard deviation of each short chain fatty acid’ concentration (μmol/g) in patients and healthy controls

SCFA (μmol/g)	HC	CD	CRC	AP	Intergroup comparisons (P value)
					HC/CD	HC/CRC	HC/AP	CD/CRC	CD/AP	CRC/AP
Acetic acid	30.9 ± 14.4	37.6 ± 17.9	15.4 ± 7.2	19.0 ± 17.2	0.254	< 0.001	0.0567	< 0.0001	0.031	0.772
Propionic acid	7.6 ± 4.3	8.3 ± 4.4	5.5 ± 2.9	7.8 ± 7.1	0.615	0.230	0.559	0.116	0.357	0.809
Butyric acid	7.1 ± 5.9	8.0 ± 5.8	6.7 ± 5.2	9.0 ± 12.4	0.401	0.756	0.636	0.502	0.357	0.629
isoButyric acid	0.7 ± 0.3	1.2 ± 0.6	1.4 ± 1.1	0.9 ± 0.9	0.008	0.116	0.889	0.831	0.357	0.187
isoValeric acid	0.5 ± 0.2	0.7 ± 0.6	1.3 ± 1.1	2.0 ± 3.9	0.273	0.032	0.802	0.115	0.590	0.460
Valeric acid	0.9 ± 0.7	1.2 ± 0.7	1.4 ± 1.0	1.1 ± 1.1	0.128	0.151	1.00	0.635	0.522	0.356
Total	47.7 ± 22.9	56.8 ± 27.4	32.5 ± 17.5	39.8 ± 39.3	0.380	0.044	0.187	0.005	0.084	0.846

P values assessed with Wilcoxon test. The intergroup comparisons were assessed using the Wilcoxon test. SCFA: Short chian fatty acid; HC: Healthy control; CD: Celiac disease; AP: Adenomatous polyposis; CRC: Colorectal cancer.

Table 7  P values of the Wald test conducted on the Dirichlet regression coefficients

	Acetic ac.	Propioni ac.	Butyric ac.	Isobutyric ac.	Isovaleric ac.	Valeric ac.
HC (Intercept)	5.56 × 10-12	4.29 × 10-5	0.00359	0.27547	0.09552	0.28869
State CD	0.145841	0.35339	0.12181	0.07597	0.16228	0.03966
State CRC	2.07 × 10-5	0.00536	0.00975	0.31373	0.42884	0.28591
State AP	0.000213	0.07797	0.06314	0.20993	0.79231	0.36639
Age	0.003016	0.92196	0.74284	0.38750	0.25510	0.19196
Sex	0.838224	0.01121	0.00118	0.00195	0.00213	0.00328

HC: Healthy control; CD: Celiac disease; AP: Adenomatous polyposis; CRC: Colorectal cancer.