The Quantitative Profiling of Oxylipins from Arachidonic Acid by LC-MS/MS in Feces at Birth 3 Days and 21 Days of Piglets

Abstract

Oxylipins (also called eicosanoids) are enzymatically or nonenzymatically generated by oxidation of arachidonic acid (ARA) and are major mediators of ARA effects in the body. Previous studies demonstrated the importance of ARA in infant growth, brain development, immune response, and health. With the developments in lipidomic methodologies, it is important for exploring more ARA-deprived oxylipins to better understand the physiological functions of ARA. The concentrations of oxylipins in feces were determined from days 3 to 21 postnatally of suckling piglets in vivo. Feces were collected at two critical time points of the suckling piglets (3d and 21d after birth) and about 48 oxylipins were analyzed by using a target metabolomics approach based on Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Here, 21 oxylipins were derived from ARA, and 11 differential oxylipins (Log2|fold change| ≥ 1.0) at birth 3d and 21d were identified. Particularly, 12-HETE was more abundant in feces at birth 3 days rather than 21 days. Considering that 12-HETE was a racemic mixture of stereoisomers containing the S and R enantiomers, we further detected the concentrations of 12(S)-HETE and 12(R)-HETE between the two time points by chiral LC-MS/MS analysis. There was no significant difference in the concentrations of 12(S)-HETE and 12(R)-HETE. It was showed that ARA - derived oxylipins might be related to the physiological changes of piglets during growing. Our results provided new information for describing the physiological changes of the piglets over the suckling period.

Keywords: 12-HETE; LC-MS/MS; arachidonic acid; chiral analysis; oxylipins; suckling piglets.

Figure 1

LC-MS/MS chromatogram of 48 oxylipins performed on a triple quadrupole employing dynamic MRM (n = 10). MRM metabolite detection multipeak diagram showed the substances that could be detected in the sample, with the mass spectrum peak of each color representing one metabolite detected.

Figure 2

Oxylipins were detected in feces of suckling piglets at birth day 3 and day 21 (n = 10). (a) Heat map hierarchical clustering of detected all metabolites in this study. Hierarchical trees were drawn based on detected metabolites in feces of suckling piglets at birth day 3 and day 21. The columns correspond to feces at 3d and 21d postnatally, while the rows represent different metabolites. (b) Principal component analysis for metabolites identified in feces.

Figure 3

Differential oxylipins were analyzed in feces between 3d and 21d postnatally (n = 10). (a) Heatmap analysis of detected 21oxylipins in this study. (b) Violin plot analysis of 21 differential 21oxylipins in this study. The x axis indicates the name of the groups, and the y axis indicates the expression quantity. (c) The bar plot of top 20 up-regulated and down-regulated oxylipins.

Figure 4

The quantitative content of ARA derived differential oxylipins in feces (n = 10). (a) the quantitative content of ARA in feces of suckling piglets at birth day 3 and day 21. (b) the absolute levels of ARA derived differential oxylipins. (c) KEGG pathway analysis of the ARA derived differential oxylipins in feces at 3 and 21 days after birth. Blue indicates that substances were detected but had no significantly change compared to these of 21d, and green indicates that the content of oxylipins was significantly down-regulated in feces from 21 days after birth.

Figure 5

Chiral LC-MS/MS analysis of 12-HETE in feces of suckling piglets (n = 10). (a) Chemical structure diagram of 12(S)-HETE and 12(R)-HETE. (b) Selected reaction monitoring (SRM) chromatograms demonstrating the resolution of 12(S)- and 12(R)-HETE in feces of suckling piglets at birth day 3 and day 21 (n = 9). (c) the quantitative content of 12(S)- and 12(R)-HETE in feces at 3d and 21d postnatally, values are the mean ± SD, n = 10. Statistical analysis was performed by Tukey tests, * p < 0.05, ** p < 0.01.