Faster Protocol for Endogenous Fatty Acid Esters of Hydroxy Fatty Acid (FAHFA) Measurements

Abstract

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous lipids with antidiabetic and anti-inflammatory activities. Interest in these lipids is due to their unique biological activites and the observation that insulin-resistant people have lower palmitic acid esters of hydroxystearic acid (PAHSA) levels, suggesting that a FAHFA deficiency may contribute to metabolic disease. Rigorous testing of this hypothesis will require the measurement of many clinical samples; however, current analytical workflows are too slow to enable samples to be analyzed quickly. Here we describe the development of a significantly faster workflow to measure FAHFAs that optimizes the fractionation and chromatography of these lipids. We can measure FAHFAs in 30 min with this new protocol versus 90 min using the older protocol with comparable performance in regioisomer detection and quantitation. We also discovered through this optimization that oleic acid esters of hydroxystearic acids (OAHSAs), another family of FAHFAs, have a much lower background signal than PAHSAs, which makes them easier to measure. Our faster workflow was able to quantify changes in PAHSAs and OAHSAs in mouse tissues and human plasma, highlighting the potential of this protocol for basic and clinical applications.

AIG1 and ADTRP are endogenous hydrolases of fatty acid esters of hydroxy fatty acids (FAHFAs) in mice

“…Samples were passed through a second SPE and eluent containing liberated FAHFAs was collected with ethyl acetate. Samples were dried and resuspended in 60-200 µl of methanol and analyzed using LC-MS method previously described (48). 2-10 µl of sample was injected to UPLC BEH C18 Column (Waters Acquity, 186002350).…”

Methodological Issues in Studying PAHSA Biology: Masking PAHSA Effects

“…We have not performed these experiments under these conditions, but when we deliver equivalent doses of 5- and 9-PAHSA, we observe similar fold increases in serum levels of each PAHSA (Syed et al, 2017, 2018). Previous reports from our group (Kolar et al, 2018; Yore et al, 2014; Zhang et al, 2016) and others (Brezinova et al, 2018) noted the possibility of a ceramide contaminant overlapping with 5-PAHSA, which could lead to artificially high estimates of 5-PAHSA. Therefore, we are concerned that the LC-MS protocols employed by Pflimlin et al are overestimating 5-PAHSA and we strongly caution other groups not to use their protocol to measure serum or tissue PAHSA levels.…”

“…The LC-MS protocol used by Pflimlin et al to measure PAHSA levels did not recapitulate any of the protocols published by three independent groups (Brezinova et al, 2018; Kolar et al, 2018; Kuda et al, 2018; Ma et al, 2015; Syed et al, 2018; Zhang et al, 2016; Yore et al, 2014) (Table S2) or more recently by an additional group (Zhu et al, 2018). Furthermore, no information is provided comparing their protocol to any of the reported procedures to validate their results.…”

Na dobre i na złe – czyli rola oddziaływania trzustki, śródbłonka i tkanki tłuszczowej w regulacji funkcjonowania komórek β i rozwoju cukrzycy typu 2 związanej z otyłością

“…Each FAHFA family consists of multiple isomers that differ in their branched ester position. Palmitic acid esters of hydroxyl stearic acid (PAHSA) were markedly decreased in subcutaneous white adipose tissue and serum in obese insulin-resistant mice and humans [59,61]. Four PAHSA isomers were confirmed in the mouse pancreas [59,62], but their effect on β-cell metabolism remains to be determined.…”