Analysis of identified sterols, bile acids, and acylcarnitines

Version 1

See “List of identified sterols, bile acids, and acylcarnitines” for details.

Figure 1: Separation of the different classes of standards (sterols, bile acids, and acylcarnitines) based on retention time in reversed-phase liquid chromatography in the positive ionization mode.

Figure 2: Extracted ion chromatograms of the base peak spectra of detected sterols acquired from reference “BACSMLS” standards in the positive ionization mode. Peak identities are as follows: (1) cortisone, (2) corticosterone, (3) 25-hydroxycholesterol, (4) 24-hydroxycholesterol, (5) 27-hydroxycholesterol, (6) diosgenin, (7) 7α-hydroxy-4-cholesten-3-one, (8) 7α-hydroxycholesterol, (9) 7β-hydroxycholesterol, (10) 7-ketocholesterol (5-cholesten-3β-ol-7-one), (11) cholesterol 5α,6α-epoxide, (12) desmosterol, (13) ergosterol, (14) 7-dehydrocholesterol, (15) brassicasterol, (16) 5β-cholestan-3α-ol (epicoprostanol), (17) lathosterol, (18) cholesterol, (19) coprostanol (coprostan-3-ol), (20) lanosterol, (21) dihydrocholesterol (5α-cholestan-3β-ol), (22) stigmasterol, (23) campesterol, (24) β-sitosterol.

Figure 3: Extracted ion chromatogram of the base peak spectra of bile acids acquired from reference “BACSMLS” standards in the positive ionization mode. (A) EICs of unconjugated bile acids. Peak identities are as follows: (1) dehydrocholic acid, (2) ursocholic acid, (3) 7-ketodeoxycholic acid (7-keto-3α,12-α-dihydroxycholanic acid), (4) 12-ketochenodeoxycholic acid ((3alpha,5beta,7alpha)-3,7-dihydroxy-12-oxocholan-24-oic acid), (5) alpha-muricholic acid, (6) beta-muricholic acid, (7) murideoxycholic acid, (8) ursodeoxycholic acid, (9) gamma-muricholic acid (hyocholic acid), (10) 7-ketochenodeoxycholic acid (nutriacholic acid), (11) hyodeoxycholic acid, (12) cholic acid, (13) A=allocholic acid, (14) nordeoxycholic acid, (15) chenodeoxycholic acid, (16) deoxycholic acid, (17) 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA), (18) coprocholic acid (3α,7α,12α-trihydroxycoprostanic acid), (19) dehydrolithocholic acid (3-oxo-5β-cholanoic acid), (20) lithocholic acid. (B) EICs of glycine-conjugated bile acids acquired from reference “BACSMLS” standards—(a) Glycoursodeoxycholic acid, (b) glycohyodeoxcholic acid, (c) glycocholic acid, (d) glycochenodeoxycholic acid, (e) glycodeoxycholic acid, (f) glycolithocholic acid. (C) EICs of taurine-conjugated bile acids—(i) tauromuricholic acid (ii) tauroursodeoxycholic acid, (iii) taurocholic acid, (iv) taurochenodesoxycholic acid, (v) taurodeoxycholic acid, (vi) taurolithocholic acid.

Figure 4: Extracted ion chromatograms of the molecular ions of detected acylcarnitines acquired from reference “BACSMLS” standard samples in the positive ionization mode. Peak identities are as follows: (1) succinylcarnitine, (2) hydroxybutyrylcarnitine (3) acetyl-DL-carnitine, (4) glutarylcarnitine, (5) propionyl-L-carnitine, (6) adipoyl-L-carnitine, (7) methylglutaryl-L-carnitine, (8) isobutyryl-L-carnitine, (9) butanoylcarnitine (Butyryl-L-carnitine), (10) tiglylcarnitine, (11) 2-methylbutyroylcarnitine, (12) isovaleryl-L-carnitine, (13) valeryl-L-carnitine, (14) hexanoylcarnitine, (15) octenoyl-L-carnitine, (16) octanoyl-L-carnitine, (17) decanoyl-L-carnitine, (18) 3-hydroxydodecanoylcarnitine, (19) dodecenoylcarnitine, (20) dodecanoylcarnitine (lauroyl-L-carnitine), (21) tetradecenoyl-L-carnitine, (22) myristoyl-L-carnitine, (23) 3-hydroxyhexadecanoylcarnitine, (24) linoleylcarnitine (cis,cis-9,12-octadecadienoyl-L-carnitine), (25) hexadecanoylcarnitine (palmitoyl-L-carnitine), (26) oleoylcarnitine, (27) octadecanoylcarnitine (stearoyl-L-carnitine).

Figure 5: Principal component analysis (PCA) of the biological samples analyzed in the positive ionization mode. This figure contains the first two principal components and their scores PC1 and PC2, which explain 24.5% and 19.9% of the variation within the data, respectively. The shapes denote the different species and the colors denote the sample types. The plot shows three clusters (statistical significance not evaluated): blue ellipse—plasma, heart, liver, and intestinal tissues; green ellipse—intestinal content and stool samples; brown ellipse—bile.

Figure 6: Correlation matrix of analyzed biological samples in the positive ionization mode. Values and shading intensities represent Pearson rank correlation coefficients.

Figure 7: An unsupervised hierarchical clustering analysis based on the qualitative analysis of “BACSMLS”. Each row shows the data for a specific sterol/bile acid/acylcarnitine, and each column shows the “BACSMLS” profiles for the different biological samples. All the identified metabolites were included. Clustering was performed for both metabolites and biological samples (dendrograms not shown for metabolites). The clustering analysis efficiently distinguishes between the different sample types and, more importantly, between the studied species. The raw peak area values from MS-DIAL were used to determine the relative levels of the compounds in the biological samples. For isomers, this value was manually integrated in the vendor software—Agilent MassHunter Qualitative Analysis B.07.00. The peak areas were normalized across all compounds and samples using the mean and the standard deviation (Z-score).