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Validated bioanalysis of oxylipins confirms specialized pro-resolving mediator formation in vitro and in vivo
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DOI：10.1016/j.jlr.2026.101075.png)
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Abstract
En 中文
Specialized pro-resolving mediators (SPM) are di- or trihydroxylated polyunsaturated fatty acids with potent inflammation-resolving features. SPM typically occur at lower concentrations than classical pro-inflammatory eicosanoids, making their detection highly challenging. Thus, reporting of biological SPM levels has been discrepant and not always transparent, sparking uncertainty concerning the formation capacity for di- and especially for trihydroxylated SPM in vitro and in vivo. Here, we create common ground by providing a systematic and comprehensive assessment of SPM formation across a broad range of commonly employed in vitro, ex vivo, and in vivo matrices. For this purpose, a quantitative UHPLC-MS/MS method targeting 72 oxylipins (including 19 SPM) was validated in accordance with recommendations by the International Lipidomics Society. By including sample preparation (solid phase extraction) in all validation processes, a conservative lower limit of quantification (10–100 pg/mL matrix) was found to meet clearly defined criteria for signal/noise, accuracy, precision, selectivity, specificity, matrix effects, recovery, and carry-over for 19 SPM. Our results offer analytically defensible reference data to demonstrate (i) the absence of relevant SPM levels in stimulated whole blood and unstimulated cell models; (ii) only trace SPM formation in stimulated peripheral blood mononuclear cells, M1-macrophages, neutrophils, and platelet incubations; but (iii) significant SPM formation in stimulated M2a-macrophages and neutrophil/platelet co-incubations. In healthy C57BL/6JRj mice, SPM formation was low yet organ-specific, with relevant amounts of dihydroxylated SPM detected in spleen. Taken together, we demonstrate SPM formation to be robust, matrix- and stimulus-dependent, but confined to specific di- and trihydroxylated SPM.
Keywords:
inflammation
cell signaling
lipoxygenase
omega-3 fatty acids
arachidonic acid
oxidized lipids
lipidomics
mass spectrometry
liquid chromatography
validation
AA
arachidonic acid
COX
cyclooxygenase
CYP
cytochrome P450
DHA
docosahexaenoic acid
EPA
eicosapentaenoic acid
EPI
enhanced product ion
ES
external standard
FWHM
full width at half maximum
HDHA
hydroxyeicosahexaenoic acid
HEPE
hydroxyeicosapentaenoic acid
HETE
hydroxyeicosatetraenoic acid
IS
internal standard
LLOQ
lower limit of quantification
LM
lipid mediator
LOD
limit of detection
LOX
lipoxygenase
LT
leukotriene
LX
lipoxin
MaR
maresin
MDM
monocyte-derived macrophage
N
noise
PD
protectin
PG
prostaglandin
QC
quality control sample
R
resolution
RP
reversed-phase
Rt
retention time
Rv
resolvin
S
signal
SIL
stable-isotope-labeled
sMRM
scheduled multiple reaction monitoring
SPE
solid phase extraction
SPM
specialized pro-resolving mediator
TX
thromboxane
ULOQ
upper limit of quantification
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