Figure 1.
CONSORT flowchart of the study AHAB-2.
Patients recruited for the present substudy are indicated at the bottom of the flow diagram under the heading “Analysis”.
Figure 2.
Generation of 7-oxo-DHA and 7-oxo-DPA by neutrophils supplemented with DHA or DPA and stimulated with calcium ionophore.
Freshly isolated neutrophils were supplemented with 30 µM DHA (A–D) or DPA (E–G) and stimulated with calcium ionophore. Samples were collected at 15 min and cell extracts were reacted with 500 mM BME for detection of electrophilic fatty acids. (A,E) Chromatograms were acquired following the neutral loss of BME using the following transitions: 419.2 to 341.2(A) and 421.2 to 343.2 (E). Insets show mass spectra with respective BME β-elimination due to in source fragmentation. (B,F) Product ion analysis of BME-oxo-DHA (m/z 419.2, B) and BME-oxo-DPA (m/z 421.2, F); (C,G) MS/MS analysis of the free oxo-DHA (C) and oxo-DPA (G) generated by in-source fragmentation upon respective BME neutral loss; (D) MS/MS analysis of 7-oxo-DHA synthetic standard; (H) chemical structure and fragmentation pattern of 7-oxo-DHA and 7-oxo-DPA. 7-oxo-DHA standard was enzymatically synthesized by incubating 20 µM 7-OH-DHA with 3α-hydroxysteroid dehydrogenase in the presence of 100 µM NAD+ for 10 min at 37°C.
Figure 3.
Generation of OH-oxo-DHA and OH-oxo-DPA by neutrophils supplemented with DHA or DPA and stimulated with calcium ionophore.
Freshly isolated neutrophils were supplemented with 30 µM DHA (A-D) or DPA (E-H) and stimulated with calcium ionophore. Samples were collected at 15 min and cell extracts were reacted with excess BME for detection of electrophilic fatty acid derivatives. (A, E) Chromatograms showing elution of ions with m/z 435.2 (A) and 437.2 (E) and their respective BME β-elimination due to in source fragmentation (insets). (B, F) Product ion analysis of BME-OH-oxo-DHA (m/z 435.2, B) and BME-OH-oxo-DPA (m/z 437.2, F); (C, G) MS/MS analysis of the free OH-oxo-DHA (C) and OH-oxo-DPA (G) generated by in-source fragmentation upon respective BME neutral loss; (D, H) proposed chemical structure and fragmentation pattern of 17-OH-7-oxo-DHA (D) and 17-OH-7-oxo-DPA (H).
Figure 4.
Time course of the generation of electrophilic 7-oxo-DHA, 7-oxo-DPA and 5-oxo-EPA by activated neutrophils.
Freshly isolated neutrophils from three independent healthy subjects were stimulated with calcium ionophore, samples were collected at the indicated time points and cell extracts were reacted with 500α,β-unsaturated omega-3-derived fatty acids by LC-MS/MS.
Figure 5.
Endogenous generation of 5-oxo-EPA, 7-oxo-DHA, 7-oxo-DPA and their hydroxy precursors by activated neutrophils.
Freshly isolated neutrophils were stimulated for 15-MS/MS in MRM mode. (A) BME adducts of 5-oxo-EPA, 7-oxo-DHA and 7-oxo-DPA were measured following loss of BME. (B) For the measurement of non-electrophilic hydroxy derivatives the following transitions were used: 317.2/299.2 (5-OH-EPA), 343.2/281.2 (7-OH-DHA), 345.2/327.2 (7-OH-DPA). Where available, chromatograms of synthetic standards are also reported. 7-oxo-DHA and 5-oxo-EPA standards were enzymatically synthesized by incubating 20 µM of OH-precursors with 3α-hydroxysteroid dehydrogenase, in presence of 100 µM NAD+ for 10 min at 37°. Insets show a magnified view of chromatograms. Std, standard.
Figure 6.
In vitro synthesis of 7-OH-DHA and 7-OH-DPA from DHA and DPA precursors by human recombinant 5-LO.
Human recombinant 5-LO was incubated with 30 µM DHA (A-C) or DPA (D-E) in the presence of 2 mM ATP at 37°C for 15 min and ion product formation was analyzed by LC-MS/MS using high resolution mass spectrometry (Velos/Orbitrap (A, D)). For 7-OH-DHA the elution profile of the synthetic standard is reported as grey line in the inset (A). (B, E) Product ion spectra, chemical structures and interpretation of fragmentation upon collision induced dissociation. (C) Mass spectrum of 7-OH-DHA chemical standard.
Table 1.
Characteristics of clinical trial study population.
Figure 7.
Dietary modulation of 5-oxo-EPA, 7-oxo-DHA and 7-oxo-DPA generation and their hydroxy precursors by neutrophils.
Formation of 5-oxo-EPA, 7-oxo-DHA, 7-oxo-DPA (A) and their hydroxy precursors (B) was measured in freshly isolated neutrophils under basal conditions and after stimulation with calcium ionophore in healthy subjects consuming either control soybean oil (white bars) or a fish oil-supplemented diet (black bars). Neutrophils were stimulated for 15 min with calcium ionophore, cell extracts were reacted with 500 mM BME and the BME-adducted electrophilic oxo-fatty acids as well as non-electrophilic hydroxy fatty acids were quantified by LC-MS/MS. Data are expressed as mean ± SE, n = 21 and 24 for control and fish oil, respectively. The Mann–Whitney U test was used for pair wise comparisons of patients groups. **, p-value<0.005.
Figure 8.
Enzymatic pathways leading to the formation of long chain PUFAs oxidized derivatives in activated neutrophils.
Compounds indicated in solid boxes are those measured in the present study. Compounds indicated in dotted boxes are those that may be formed in activated neutrophils but were not measured in the present study. MK-886 is the 5-LO activating protein (FLAP) inhibitor used in this study. (5-LO, 5-lipoxygenase; 5-HEDH, 5-hydroxyeicosanoid dehydrogenase).