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Activated platelets and monocytes generate four hydroxyphosphatidylethanolamines via lipoxygenase.

2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês

10.1074/jbc.a611776200

1083-351X

Benjamin H. Maskrey, Alexandra Bermúdez-Fajardo, Alwena H. Morgan, Esther Stewart-Jones, Vincent Dioszeghy, Graham W. Taylor, Paul R.S. Baker, Barbara Coles, Marcus J. Coffey, Hartmut Kühn, Valerie B. O’Donnell,

Cholesterol and Lipid Metabolism

VOLUME 282 (2007) PAGES 20151–20163 In our article, we stated, “Comparison of levels of free 15-HETE after hydrolysis of either total lipid extract (Fig. 1) or purified monocyte PE (Fig. 3) shows that the PE-esterified HETE does not seem to account for all the esterified HETE formed. Specifically, total esterified HETE varied from 100 to 2000 ng/4 × 106 cells (due to donor variation), but only 17.5 ng/4 × 106 cells was detected after purification and hydrolysis of PE from one donor. This suggests that 15-HETE is esterified into additional nonphospholipid pools that have not been detected here, although perhaps not being ionized in negative mode.” Since this work was published, we succeeded in generating an 18:0a/15-HETE-PE standard and have now been able to directly quantitate the four PE-HETEs using this lipid. Although there may be small differences in ionization efficiency between the four lipids, we believe that quantitation using these standards with liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a more accurate approach than that used in this work. Specifically, we previously purified total PE, followed by hydrolysis to liberate 15-HETE, re-extraction, and then quantitation using LC/MS/MS. We believe that these additional steps resulted in losses of lipid and significant underestimation of total levels of PE-esterified HETE. Using the new approach, our estimates of PE-HETE are now much closer to our measurements of total esterified HETE in activated monocytes (Table 1). This suggests instead that PE-HETEs are the major source of esterified HETE and that only a small amount remains that is esterified elsewhere.TABLE 1Esterified HETEs measured using LC/MS/MS in lipid extracts from ionophore-actived monocytesMoleculeControlActivatedng/4 × 106 cellsng/4 × 106 cellsFree 15-HETE (measured using LC/MS/MS)1.43257Total esterified 15-HETE (lipid extracts hydrolyzed and total esterified 15-HETE measured using LC/MS/MS)11.116416:0p/15-HETE-PE (measured using synthetic standard)3.863.818:0p/15-HETE-PE (measured using synthetic standard)1.320.318:1p/15-HETE-PE (measured using synthetic standard)2.737.718:0a/15-HETE-PE (measured using synthetic standards)1.629Total PE-HETEs (totaled from individual species measured above)9.6150.8 Open table in a new tab