Identification of short‐chain fatty acid esters of hydroxy fatty acids (SFAHFAs) in a murine model by nontargeted analysis using ultra‐high‐performance liquid chromatography/linear ion trap quadrupole‐Orbitrap mass spectrometry

Abstract

RationaleFatty acid esters of hydroxy fatty acids (FAHFAs) are recently discovered endogenous lipids with outstanding health benefits. FAHFAs are known to exhibit antioxidant, antidiabetic and anti‐inflammatory properties. The number of known long‐chain FAHFAs in mammalian tissues and dietary resources increased recently because of the latest developments in high‐resolution tandem mass spectrometry techniques. However, there are no reports on the identification of short‐chain fatty acid esterified hydroxy fatty acids (SFAHFAs).MethodsIntestinal contents, tissues, and plasma of rats fed with high‐fat diet (HFD) and normal diet (ND) were analyzed for fatty acids, hydroxy fatty acids, and FAHFAs using ultra‐high‐performance liquid chromatography (UHPLC) and linear trap quadrupole‐Orbitrap mass spectrometry (LTQ Orbitrap MS) with negative heated electrospray ionization.ResultsUntargeted analysis of total lipid extracts from murine samples (male 13‐week‐old WKAH/HKmSlc rats) led to the identification of several new SFAHFAs of acetic acid or propanoic acid esterified long‐chain (>C20)‐hydroxy fatty acids. Furthermore, MS3 analysis revealed the position of the hydroxyl group in the long‐chain fatty acid as C‐2. The relative amounts of SFAHFAs were quantified in intestinal contents and their tissues (Cecum, small intestine, and large intestine), liver, and plasma of rats fed with HFD and ND. The large intestine showed the highest abundance of SFAHFAs with a concentration range from 0.84 to 57 pmol/mg followed by the cecum with a range of 0.66 to 28.6 pmol/mg. The SFAHFAs were significantly altered between the HFD and ND groups, with a strong decreasing tendency under HFD conditions.ConclusionsIdentification of these novel SFAHFAs can contribute to a better understanding of the chemical and biological properties of individual SFAHFAs and their possible sources in the gut, which in turn helps us tackle the role of these lipids in various metabolic diseases.

Chemical Labeling Assisted Detection and Identification of Short Chain Fatty Acid Esters of Hydroxy Fatty Acid in Rat Colon and Cecum Contents

“…So far, the FAHFAs identified are esterified at carbon chain≥C5 [ 5 , 10 ], but esters at C2 position are limited [ 11 ]. In our previous study, we have confirmed that the location of the hydroxy group is at C-2 of long-chain fatty acid by MS n analysis [ 12 ]. Further successive loss of neutral molecules of CH2=CO ( m / z 42 Da) and H 2 O ( m / z 18 Da) from the fragment ion m / z 396 gives a molecular ion at m / z 354 and m / z 336, respectively.…”

“…As the data shows, signal intensity of DMED-SFAHFA is increased by almost three folds higher compared to unlabeled SFAHFA. Though this change is small, it contributes largely to the detection of other minor SFAHFAs, which were unidentified in our previous study [ 12 ]. Table 1 shows the list of SFAHFAs identified after DMED labeling, which includes additional six new molecular species which were identified in this study.…”

“…Alternatively, the loss of CH 3 COOH ( m / z 60 Da) from the molecular ion confirms the acyl side chain of hydroxy fatty acid is acetate. Moreover, acetic acid esterified compounds generally produce a fragment ion by the loss of a neutral molecule of acetic acid by CID, which is evident in the literature [ 12 , 18 ].…”

“…A variety of regio-isomers have been identified with an esterified hydroxyl group position at 5, 7, 8, 9, 10, 11, 12, and 13 [ 5 , 10 ], but there are limited reports on C2 or C3 [ 11 ] although C2 and C3 hydroxy fatty acids are widely spread in nature. We have recently identified a new type of lipids to the FAHFAs family named short-chain fatty acid esters of hydroxyl fatty acids (SFAHFAs) with ester linkage at C2 in the various tissues, plasma, and contents of the gastrointestinal tract [ 12 ]. Several liquid-chromatography mass spectrometry (LC-MS) based analytical methods were established to determine FAHFAs in foods and various biological matrices [ 5 , 6 , 10 , 13 , 14 ].…”

“…In our previous study, we have uncovered about ten new SFAHFAs, excluding some having unseparated structural isomers by untargeted LC-MS analysis with MS n measurements [ 12 ]. These molecular species are low in abundance, but were significantly altered with the high-fat diet in the murine model.…”