Faster Protocol for Endogenous Fatty Acid Esters of Hydroxy Fatty Acid (FAHFA) Measurements - PubMed (original) (raw)

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

Matthew J Kolar et al. Anal Chem. 2018.

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.

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Figures

Figure 1. Development of a faster FAHFA analysis method

(a) Structures of 5-, 9-, and 12-PAHSA and 12-OAHSA. Extracted ion chromatograms showing the retention times of 5-, 9-, and 12-PAHSA and 5-, 9-, 12-OAHSA standards on a Luna C18(2) column (3 µm, 250 × 2.0 mm, Phenomenex) (b) and an Acquity UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm, Waters) (c).

Figure 2. Resolution of 5-PAHSA and ceramide

(a) Traces showing the effect of altering column temperature ranging from 20 °C to 40 °C on resolution of 5-PAHSA and the ceramide. (b) Traces showing the effect of different flow rates on the resolution of 5-PAHSA and ceramide. (c) Traces showing the effect of increasing the mobile phase pH. Black lines represent the transition m/z 537 → 255 (which is shared by PAHSAs and the ceramide contaminant). Purple line represents the internal standard 13C16-5-PAHSA, which is used as a surrogate for analyzing 5-PAHSA retention time. Although 5-PAHSA is present in mouse serum, it is not shown on the chromatogram because it needs magnification to be seen.

Figure 3. Stability of PAHSAs with different NH4OH percentages in mobile phase

Extracted ion chromatogram of 12-, 10-, and 9-PAHSA with their respective AUC with either 0.01% NH4OH (top trace) or 0.03% NH4OH (bottom trace).

Figure 4. SPE cartridges have variable PAHSA background from lot-to-lot

Extracted ion chromatograms for PAHSAs (top) and internal standard 13C4-9-PAHSA (bottom). Internal standard was added during the sample loading step to account for variation in the SPE procedure.

Figure 5. Background SPE signal for OAHSAs and PAHSAs

Extracted ion chromatograms for background PAHSAs (red) and OAHSAs (blue) in SPE cartridges from two different lots.

Figure 6. Effects of prewashing SPE cartridge on PAHSA background

SPE columns from the same lot number were prewashed with EA alone, MeOH alone, EA + MeOH, or EA + MeOH + DCM and their PAHSA background was compared to PAHSA background from a non-prewashed column (black line).

Figure 7. Analysis of OAHSAs and PAHSAs in PGWAT of WT and AG4OX mice with our original and shorter method

Extracted ion chromatograms comparing PGWAT PAHSAs in AG4OX and WT mice using the original (a) and shorter method (b). Extracted ion chromatograms analyzing OAHSAs in PGWAT WT and AG4OX mice in the original (c) and shorter method (d).

Figure 8. PAHSA and OAHSA quantification and distribution in WT and AG4OX mice PGWAT

Quantification of PAHSA (a) and OAHSA (b) isomers in AG4OX and WT mice PGWAT. Data are expressed as means ± SEM. Differences between groups was evaluated with Mann-Whitney test. n=3–5/group, *p<0.05.

Figure 9. PAHSA and OAHSA regioisomers in fasting human plasma

Extracted ion chromatogram of PAHSAs (top trace) and OAHSAs (bottom trace).

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Faster Protocol for Endogenous Fatty Acid Esters of Hydroxy Fatty Acid (FAHFA) Measurements - PubMed (original) (raw)