Analysis of HETEs in human whole blood by chiral UHPLC-ECAPCI/HRMS

Abstract

The biosynthesis of eicosanoids occurs enzymatically via lipoxygenases, cyclooxygenases, and cytochrome P450, or through nonenzymatic free radical reactions. The enzymatic routes are highly enantiospecific. Chiral separation and high-sensitivity detection methods are required to differentiate and quantify enantioselective HETEs in complex biological fluids. We report here a targeted chiral lipidomics analysis of human blood using ultra-HPLC-electron capture (EC) atmospheric pressure chemical ionization/high-resolution MS. Monitoring the high-resolution ions formed by the fragmentation of pentafluorobenzyl derivatives of oxidized lipids during the dissociative EC, followed by in-trap fragmentation, increased sensitivity by an order of magnitude when compared with the unit resolution MS. The 12(S)-HETE, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid [12(S)-HHT], and 15(S)-HETE were the major hydroxylated nonesterified chiral lipids in serum. Stimulation of whole blood with zymosan and lipopolysaccharide (LPS) resulted in stimulus- and time-dependent effects. An acute exposure to zymosan induced ∼80% of the chiral plasma lipids, including 12(S)-HHT, 5(S)-HETE, 15(R)-HETE, and 15(S)-HETE, while a maximum response to LPS was achieved after a long-term stimulation. The reported method allows for a rapid quantification with high sensitivity and specificity of enantiospecific responses to in vitro stimulation or coagulation of human blood.

Trial registration: ClinicalTrials.gov NCT02095288.

Keywords: chiral hydroxyeicosatetraenoic acids; coagulation; human blood; hydroxyeicosatetraenoic acids; plasma lipidomics; serum lipidomics; ultra-high-performance liquid chromatography-electron capture atmospheric pressure chemical ionization high-resolution mass spectrometry.

Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.

Figures

Fig. 1.

ECAPCI-MS/HRMS of 15-HETE-PFB. The low energy electrons generated from corona discharge interact with the nitrogen sheath gas and generate radical cations. Dissociative EC results in a very strong product ion corresponding to 15-HETE anion m/z 319.2269.

Fig. 2.

Product spectra from ECAPCI-MS/HRMS of different HETE-PFBs. Collision-induced dissociation of the parent [HETE-PFB]− generates specific product ions for each stereoisomer. 5-HETE (A), 11-HETE (B), 12-HETE (C), and 20-HETE (D).

Fig. 3.

Typical LC-ECAPCI-MS/HRMS chromatograms of HETEs as PFB derivatives. A: Extracted from 0.2 ml human serum and spiked with a synthetic heavy isotope analog internal standard {1 ng of [2H8]-15(S)-HETE}. B: Extracted from 0.2 ml human plasma and spiked with a synthetic heavy isotope analog internal standard {1 ng of [2H8]-15(S)-HETE}.

Fig. 4.

Enantioselective formation of HETEs and Tx during the intrinsic pathway of coagulation of human blood. Human whole blood was incubated at 37°C for 1 h and serum was removed for analysis of TxB2 and HETEs, as described in the Materials and Methods. Lipids from serum were compared with plasma lipids from untreated whole blood. A, B: Data are expressed as nanograms of lipid per sample volume and represent the mean ± SEM; ##P ≤ 0.01, ###P ≤ 0.001, ####P ≤ 0.0001 between enantiomers for serum; paired t-test; n = 9. ND, not detected; ns, not significant. Shaded areas depict interquartile range of distribution of the lipid concentrations. Absolute quantities of lipids are summarized in Table 1. C: PCA of serum and plasma lipids presented in a three-dimensional loading plot. Each symbol represents an independent subject (n = 9). Ellipses denote 95% confidence regions. Two-way ANOVA of the means of serum versus plasma for all analytes, P = 2 × 10−16.

Fig. 5.

Time- and stimulus-dependent effects of in vitro stimulation on chiral HETEs in human whole blood. Heparinized human whole blood was stimulated with 100 μg/ml LPS and 125 μg/ml zymosan (Zym), alone (n = 15) or in combination (L+Z, n = 5) for 4 and 24 h at 37°C. Plasma was removed for analysis of chiral HETEs as described in the Materials and methods, and significant fold differences over the vehicle (PBS) control were summarized on a log scale in a heat map (A). Red and blue boxes denote elevation and reduction, respectively. Unpaired two-tailed t-test, n = 5–15. Absolute quantities of lipids are summarized in Table 2. Effects of stimulating conditions on lipid production at 4 h (B) and 24 h (C). Shaded areas depict interquartile range of distribution of the lipid concentrations. D: PCA of plasma lipids after stimulation of whole blood for 4 h and 24 h and presented in three-dimentional loading plots. Each symbol represents an independent subject (n = 5–15). E: Venn diagrams showing the number of analytes common or unique to treatments after 4 h and 24 h of stimulation, relative to PBS control. The percentage of total analytes affected by a specific treatment is shown in boxes.

Fig. 6.

Enantioselective biosynthesis of 15-HETE compared with nonenzymatic formation of 9-HETE in stimulated human blood. Heparinized human whole blood was stimulated with 100 μg/ml LPS and 125 μg/ml zymosan (Zym), alone (n = 15) or in combination (LPS+Zym, n = 5), for 4 and 24 h at 37°C. Plasma was removed for analysis of 15-HETE (A) and 9-HETE (B) as described in the Materials and Methods. Data are expressed as mean ± SEM; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 versus PBS; one-way ANOVA, Dunnett’s test; n = 5–15. ns, not significant.