BACKGROUND: Oxidized derivatives of polyunsaturated fatty acids, collectively known as oxylipins, are labile bioactive mediators with diverse roles in human physiology and pathology. Oxylipins are increasingly being measured in plasma collected in clinical studies to investigate biological mechanisms and as pharmacodynamic biomarkers for nutrient-based and drug-based interventions. Whole blood is generally stored either on ice or at room temperature prior to processing. However, the potential impacts of delays in processing, and of temperature prior to processing, on oxylipin concentrations are incompletely understood. OBJECTIVE: To evaluate the effects of delayed processing of blood samples in a timeframe that is typical of a clinical laboratory setting, using typical storage temperatures, on concentrations of representative unesterified oxylipins measured by liquid chromatography-tandem mass spectrometry. DESIGN: Whole blood (drawn on three separate occasions from a single person) was collected into 5 mL purple-top potassium-EDTA tubes and stored for 0, 10, 20, 30, 60 or 120 min at room temperature or on wet ice, followed by centrifugation at 4 °C for 10 min with plasma collection. Each sample was run in duplicate, therefore there were six tubes and up to six data points at each time point for each oxylipin at each condition (ice/room temperature). Representative oxylipins derived from arachidonic acid, docosahexaenoic acid, and linoleic acid were quantified by liquid chromatography tandem mass spectrometry. Longitudinal models were used to estimate differences between temperature groups 2 h after blood draw. RESULTS: We found that most oxylipins measured in human plasma in traditional potassium-EDTA tubes are reasonably stable when stored on ice for up to 2 h prior to processing, with little evidence of auto-oxidation in either condition. By contrast, in whole blood stored at room temperature, substantial time-dependent increases in the 12-lipoxygenase-derived (12-HETE, 14-HDHA) and platelet-derived (thromboxane B2) oxylipins were observed. CONCLUSION: These findings suggest that certain plasma oxylipins can be measured with reasonable accuracy despite delayed processing for up to 2 h when blood is stored on ice prior to centrifugation. 12-Lipoxygenase- and platelet-derived oxylipins may be particularly sensitive to post-collection artifact with delayed processing at room temperature. Future studies are needed to determine impacts of duration and temperature of centrifugation on oxylipin concentrations.

Background

Oxidized derivatives of polyunsaturated fatty acids, collectively known as oxylipins, are labile bioactive mediators with diverse roles in human physiology and pathology. Oxylipins are increasingly being measured in plasma collected in clinical studies to investigate biological mechanisms and as pharmacodynamic biomarkers for nutrient-based and drug-based interventions. Whole blood is generally stored either on ice or at room temperature prior to processing. However, the potential impacts of delays in processing, and of temperature prior to processing, on oxylipin concentrations are incompletely understood.

Objective

To evaluate the effects of delayed processing of blood samples in a timeframe that is typical of a clinical laboratory setting, using typical storage temperatures, on concentrations of representative unesterified oxylipins measured by liquid chromatography-tandem mass spectrometry.

Design

Whole blood (drawn on three separate occasions from a single person) was collected into 5 mL purple-top potassium-EDTA tubes and stored for 0, 10, 20, 30, 60 or 120 min at room temperature or on wet ice, followed by centrifugation at 4 °C for 10 min with plasma collection. Each sample was run in duplicate, therefore there were six tubes and up to six data points at each time point for each oxylipin at each condition (ice/room temperature). Representative oxylipins derived from arachidonic acid, docosahexaenoic acid, and linoleic acid were quantified by liquid chromatography tandem mass spectrometry. Longitudinal models were used to estimate differences between temperature groups 2 h after blood draw.

Results

We found that most oxylipins measured in human plasma in traditional potassium-EDTA tubes are reasonably stable when stored on ice for up to 2 h prior to processing, with little evidence of auto-oxidation in either condition. By contrast, in whole blood stored at room temperature, substantial time-dependent increases in the 12-lipoxygenase-derived (12-HETE, 14-HDHA) and platelet-derived (thromboxane B2) oxylipins were observed.

Conclusion

These findings suggest that certain plasma oxylipins can be measured with reasonable accuracy despite delayed processing for up to 2 h when blood is stored on ice prior to centrifugation. 12-Lipoxygenase- and platelet-derived oxylipins may be particularly sensitive to post-collection artifact with delayed processing at room temperature. Future studies are needed to determine impacts of duration and temperature of centrifugation on oxylipin concentrations.

Oxidized lipids derived from omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids, collectively known as oxylipins, are bioactive signaling molecules that play diverse roles in human health and disease. Supplementation with n-3 docosahexaenoic acid (DHA) during pregnancy has been reported to decrease the risk of preterm birth in singleton pregnancies, which may be due to effects of DHA supplementation on oxylipins or their precursor n-6 and n-3 fatty acids. There is only limited understanding of the levels and trajectory of changes in plasma oxylipins during pregnancy, effects of DHA supplementation on oxylipins and unesterified fatty acids, and whether and how oxylipins and their unesterified precursor fatty acids influence preterm birth. In the present study we used liquid chromatography-tandem mass spectrometry to profile oxylipins and their precursor fatty acids in the unesterified pool using plasma samples collected from a subset of pregnant Australian women who participated in the ORIP (Omega-3 fats to Reduce the Incidence of Prematurity) study. ORIP is a large randomized controlled trial testing whether daily supplementation with n-3 DHA can reduce the incidence of early preterm birth compared to control. Plasma was collected at study entry (≈pregnancy week 14) and again at ≈week 24, in a subgroup of 48 ORIP participants-12 cases with spontaneous preterm (<37 weeks) birth and 36 matched controls with spontaneous term (≥40 weeks) birth. In the combined preterm and term pregnancies, we observed that in the control group without DHA supplementation unesterified AA and AA-derived oxylipins 12-HETE, 15-HETE and TXB2 declined between weeks 14-24 of pregnancy. Compared to control, DHA supplementation increased unesterified DHA, EPA, and AA, DHA-derived 4-HDHA, 10-HDHA and 19,20-EpDPA, and AA-derived 12-HETE at 24 weeks. In exploratory analysis independent of DHA supplementation, participants with concentrations above the median for 5-lipoxygenase derivatives of AA (5-HETE, Odds Ratio (OR) 8.2; p = 0.014) or DHA (4-HDHA, OR 8.0; p = 0.015) at 14 weeks, or unesterified AA (OR 5.1; p = 0.038) at 24 weeks had higher risk of spontaneous preterm birth. The hypothesis that 5-lipoxygenase-derived oxylipins and unesterified AA could serve as mechanism-based biomarkers predicting spontaneous preterm birth should be evaluated in larger, adequately powered studies.

Oxidized lipids derived from omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids, collectively known as oxylipins, are bioactive signaling molecules that play diverse roles in human health and disease. Supplementation with n-3 docosahexaenoic acid (DHA) during pregnancy has been reported to decrease the risk of preterm birth in singleton pregnancies, which may be due to effects of DHA supplementation on oxylipins or their precursor n-6 and n-3 fatty acids. There is only limited understanding of the levels and trajectory of changes in plasma oxylipins during pregnancy, effects of DHA supplementation on oxylipins and unesterified fatty acids, and whether and how oxylipins and their unesterified precursor fatty acids influence preterm birth. In the present study we used liquid chromatography-tandem mass spectrometry to profile oxylipins and their precursor fatty acids in the unesterified pool using plasma samples collected from a subset of pregnant Australian women who participated in the ORIP (Omega-3 fats to Reduce the Incidence of Prematurity) study. ORIP is a large randomized controlled trial testing whether daily supplementation with n-3 DHA can reduce the incidence of early preterm birth compared to control. Plasma was collected at study entry (≈pregnancy week 14) and again at ≈week 24, in a subgroup of 48 ORIP participants-12 cases with spontaneous preterm (<37 weeks) birth and 36 matched controls with spontaneous term (≥40 weeks) birth. In the combined preterm and term pregnancies, we observed that in the control group without DHA supplementation unesterified AA and AA-derived oxylipins 12-HETE, 15-HETE and TXB2 declined between weeks 14-24 of pregnancy. Compared to control, DHA supplementation increased unesterified DHA, EPA, and AA, DHA-derived 4-HDHA, 10-HDHA and 19,20-EpDPA, and AA-derived 12-HETE at 24 weeks. In exploratory analysis independent of DHA supplementation, participants with concentrations above the median for 5-lipoxygenase derivatives of AA (5-HETE, Odds Ratio (OR) 8.2; p = 0.014) or DHA (4-HDHA, OR 8.0; p = 0.015) at 14 weeks, or unesterified AA (OR 5.1; p = 0.038) at 24 weeks had higher risk of spontaneous preterm birth. The hypothesis that 5-lipoxygenase-derived oxylipins and unesterified AA could serve as mechanism-based biomarkers predicting spontaneous preterm birth should be evaluated in larger, adequately powered studies.

Background

Linoleic acid (LA) is abundant in modern industrialized diets. Oxidized LA metabolites (OXLAMs) and reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), are present in heated vegetable oils and can be endogenously synthesized following consumption of dietary LA. OXLAMs have been implicated in cerebellar degeneration in chicks; 4-HNE is linked to neurodegenerative conditions in mammals. It unknown whether increasing dietary LA or OXLAMs alters the levels of oxidized fatty acids (oxylipins), precursor fatty acids, or 4-HNE in mammalian brain.

Objectives

To determine the effects of increases in dietary OXLAMs and dietary LA, on levels of fatty acids, oxylipins, and 4-HNE in mouse brain tissues.

Methods

Mice (n = 8 per group) were fed one of three controlled diets for 8 weeks: (1) a low LA diet, (2) a high LA diet, or (3) the low LA diet with added OXLAMs. Brain fatty acids, oxylipins, and 4-HNE were quantified in mouse cerebellum and cerebral cortex by gas chromatography-flame ionization detection, liquid chromatography-tandem mass spectrometry, and immunoblot, respectively.

Results

Increasing dietary LA significantly increased omega-6 fatty acids, decreased omega-3 fatty acids, and increased OXLAMs in brain. Dietary OXLAMs had minimal effect on oxidized lipids but did decrease both omega-6 and omega-3 fatty acids. Neither dietary LA nor OXLAMs altered 4-HNE levels.

Conclusion

Brain fatty acids are modulated by both dietary LA and OXLAMs, while brain OXLAMs are regulated by endogenous synthesis from LA, rather than incorporation of preformed OXLAMs.